Prodrugs of prostate specific membrane antigen (PSMA) inhibitor

ABSTRACT

Methods and compounds are disclosed for treating a disease or condition by inhibiting PSMA (Prostate Specific Membrane Antigen) using prodrugs of 2-PMPA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/968,074, filed May 1, 2018, which is a divisional of U.S. patentapplication Ser. No. 15/502,105, filed Feb. 6, 2017, now U.S. Pat. No.9,988,407, which is a § 371 U.S. National Entry of PCT/US2015/044053,filed Aug. 6, 2015, which claims the benefit of U.S. ProvisionalApplication No. 62/033,926, filed Aug. 6, 2014, which is incorporatedherein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA161056 awardedby the National Cancer Institute (NCI). The government has certainrights in the invention.

BACKGROUND

The prodrug approach is a well-established strategy to improvephysicochemical, biopharmaceutic and pharmacokinetic properties ofpotential drug molecules. Approximately 5-7% of drugs approved worldwideare prodrugs with annual sales in 2013 of $11.2 billion. Most prodrugsare simple chemical derivatives of the original molecule. Esterprodrugs, the most common prodrugs, constitute 49% of all marketedprodrugs. Reasons for the popularity of ester prodrugs include theirgenerally straight forward synthesis, their improved lipophilicity andmembrane permeability, and the ubiquitousness of estereases. An exampleof an approach to make an ester prodrug is capping the acidicmoiety(ies) with lipophilic alkyl or alkyloxymethyl esters (i.e.,pivaloyloxymethyl (POM) or propyloxycarbonyloxymethyl (POC); e.g.,Enalapril, Adefovir). Another approach is to cap the acidic moiety(ies)with amino acids to make amides that are recognizable by transporters,such as Peptide transporter 1 (PEPT1) (e.g., Pomaglumetad methionil,Valacyclovir).

PSMA (Prostate Specific Membrane Antigen), also termed GCPII (glutamatecarboxypeptidase II) and FOLH1, is a metallopeptidase that catalyzes thehydrolysis of N-acetylated aspartate-glutamate (NAAG) to N-acetylaspartate (NAA) and glutamate and cleaves terminal glutamate moietiessequentially from folate polyglutamate (Ristau et al., 2013; Mesters etal., 2006; Slusher et al., 2013). One of the most potent, selective, andefficacious PSMA inhibitors is 2-PMPA (K_(i) or IC₅₀=300 μM). After50-100 mg/kg intraperitoneal injection (i.p.) doses, it achieves 30-50μM concentrations in the brain and provides efficacy in over 20 animalmodels of the central nervous system (CNS) or peripheral nervous system(PNS) including diabetic neuropathy, peripheral neuropathy, neuropathicpain, general pain, stroke, drug addiction, amyotrophic lateralsclerosis (ALS), multiple sclerosis (MS), schizophrenia, epilepsy andseveral others associated with pathological increase of glutamateconcentration leading to excito-toxic effects and neuronal death.However, 2-PMPA is a highly polar compound with multiple carboxylatesand a zinc binding group and it has negligible oral availability.Therefore, in most cases, it must be dosed intravenously,intraperitoneally, or locally to achieve the desired effects. This factlimits its potential use as a drug since most of the above disordersrequire long term dosing for which the oral route is strongly preferred.

SUMMARY

In some aspects, the presently disclosed subject matter provides acompound of formula (I) or formula (II):

-   -   wherein:    -   each R₁, R₂, R₃, and R₄ is independently selected from the group        consisting of H, alkyl, Ar, —(CR₅R₆)_(n)—Ar,        —(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,        —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,        —(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇,        —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇,        —(CR₅R₆)_(n)—NR₈R₉, and —(CR₅R₆)_(n)—C(═O)—NR₈R₉;    -   wherein:    -   n is an integer from 1 to 20;    -   m is an integer from 1 to 20;    -   each R_(3′) and R_(4′) are independently H or alkyl;    -   each R₅ and R₆ is independently selected from the group        consisting of H, alkyl, and alkylaryl;    -   each R₇ is independently straightchain or branched alkyl;    -   Ar is aryl, substituted aryl, heteroaryl or substituted        heteroaryl; and    -   R₈ and R₉ are each independently H or alkyl; and    -   pharmaceutically acceptable salts thereof.

In particular aspects, the compound of formula (I) is selected from thegroup consisting of:

In other aspects, the presently disclosed subject matter provides amethod for treating a disease or a condition, the method comprisingadministering to a subject in need of treatment thereof, a compound offormula (I), a compound of formula (II), or a pharmaceutical compositionthereof, in an amount effective for treating the disease or condition.

In particular aspects, the disease or condition is selected from thegroup consisting of a neurodegenerative disease, multiple sclerosis(MS), cancer, angiogenesis, and inflammatory bowel disease.

In certain aspects, the neurodegenerative disease is selected from thegroup consisting of amyotrophic lateral sclerosis (ALS), Parkinson'sdisease (PD), Alzheimer's disease (AD), Huntington's disease, dementiawith Lewy Bodies (DLB), schizophrenia, pain, epilepsy, stroke, andtraumatic brain injury (TBI).

In some aspects, the disease or condition results in excess PSMAactivity. In such aspects, the method further comprises inhibiting theexcess PSMA activity when the compound of formula (I), the compound offormula (II), or a pharmaceutical composition thereof, is administered.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1 shows an example of the synthesis of Tris-POC (JAM0186);

FIG. 2 shows in vivo bioanalysis via method 1 (2-PMPA+metabolites) andmethod 2 (2-PMPA selective);

FIG. 3 shows an embodiment of a representative screening paradigm;

FIG. 4 shows an in vitro metabolic stability screen of compound 1 inhuman and mouse plasma and liver subcellular fractions;

FIG. 5 shows an in vitro metabolic stability screen of compound 6 inmouse plasma and liver subcellular fractions;

FIG. 6 shows in vivo single time point pharmacokinetic studies ofcompounds 6, 7, and 8 in mice at 30 mg/kg equivalent 2-PMPA showing30-50 fold enhancement in permeability;

FIG. 7 shows in vivo pharmacokinetic studies of compounds JHU 2109, JHU2110, and JHU 2201 in mice (method 1), showing a greater then 50-foldincrease of POM and POC prodrugs/metabolites following oral dosing;

FIG. 8 shows in vivo pharmacokinetic studies of compounds JHU 2109, JHU2110, and JHU 2201 (method 2), indicating that POM and POC esterprodrugs do not release 2-PMPA because the methyl ester is too stable;

FIG. 9A and FIG. 9B show: (FIG. 9A) in vitro metabolic stability screensof compounds JHU 2236 and JHU 2237 in mouse plasma and liver subcellularfractions (method 1); (FIG. 9B) in vivo pharmacokinetic studies ofcompounds JHU 2236, JHU 2237, JHU 2263, JHU 2264, and JHU 2265 (methods1 (total prodrug exposure) and 2 (2-PMPA release)), indicating thatincreasing ester chain length on carboxylates did not increase 2-PMPArelease and no or minimal 2-PMPA release was observed with ethyl andpropyl ester;

FIG. 10A and FIG. 10B show: (FIG. 10A) an in vivo single time pointpharmacokinetic study of Tris-POM (compound JAM0168) in mice, indicatingPOM esters on carboxylate increases 2-PMPA approximately 18-foldfollowing oral dosing; and (FIG. 10B) an in vivo single time pointpharmacokinetic study of Tris-POM (compound JAM0168) in mice (11.69mg/kg (equiv 2-PMPA); 30 min; N=3);

FIG. 11A and FIG. 11B show an in vitro metabolic stability screen ofTris-POC (compound JAM0186) in: (FIG. 11A) human and mouse plasma andliver subcellular fractions; and (FIG. 11B) human, dog, and monkeyplasma and liver subcellular fractions;

FIG. 12 shows a single dose pharmacokinetic study in mice showing plasma2-PMPA concentrations following 30 mg/kg per oral administration ofTris-POC (JAM0186; black circles) or 2-PMPA (red squares) (30 mg/kg(equiv 2-PMPA); 30 min; N=3);

FIG. 13 shows an in vitro metabolic stability screen of Tris-POC(JAM0186) in human, dog, and monkey plasma and liver subcellularfractions;

FIG. 14 shows a single dose in vivo full time course pharmacokineticstudy of Tris-POC (JAM0186) in dogs (10 mg/kg (equiv 2-PMPA); N=1)showing a high Cmax;

FIG. 15 shows an in vitro metabolic stability screen of Tris-POC(JAM0186) and Tris-methyl-POC in human, dog, and monkey plasma and liversubcellular fractions;

FIG. 16 shows a marked increase of PSMA expression in the villousepithelium from ileal sample of CD patient (Zhang et al., 2012).Immunohistochemical localization of PSMA (indicated by arrows) indiseased ileal mucosa from the proximal margin of resected ileum from anileal CD subject right panel) and a control non-IBD subject.Magnification is 100×. Bar is 200 mm;

FIG. 17 shows a marked elevation of PSMA functional enzymatic activityin the inflamed (diseased) intestinal mucosa of patients with IBD. PSMAactivity was measured from mucosa specimens (n=32) from diseased(inflamed with active disease) and normal/uninvolved (macroscopicallynormal) mucosa from IBD patients or from non-IBD controls (healthycontrols or patients with diverticulitis]. Note: PSMA is also highlyupregulated in colon cancer;

FIG. 18 shows that PSMA inhibitor (PSMAi) ameliorates DNBS-inducedcolitis as effective as sulfasalazine (Sulfs), an IBD drug beingcurrently used in the clinic. Mice receiving DNBS to induce colitis weretreated simultaneously with either Sulfs, or PSMAi (100 mg/kg). Colonweight/body weight ratio, which positively correlated with the diseaseactivity, was used as a measure for clinical activity;

FIG. 19 shows that PSMAi (2-PMPA) also ameliorates disease activity inDSS-induced murine model of colitis. C57/B6 mice (approximately 8 weeksold) that were induced to develop colitis with DSS (2.5%, 7 days indrinking water) were treated simultaneously with the vehicle or 2-PMPA(100 mg/kg), respectively. Disease activity index (DAI), whichpositively correlated with the disease severity, was used as a measurefor clinical activity. *P<0.05;

FIG. 20 shows that PSMAi (2-PMPA) effectively suppresses PSMA activityin the colonic or cecal mucosa of DSS-induced murine model of colitis.PSMA activity was measured using extract from mucosa;

FIG. 21A, FIG. 21B, FIG. 21C and FIG. 21D show that PSMAi (2-PMPA)treatment leads to not only improvement of disease but even retractionof prolapse in IL-10 knockout (IL-10KO) mice that spontaneously developcolitis: (FIG. 21A) improvement of prolapse and colonic macroscopicdisease (inflammation, hypertrophy, stool inconsistency); (FIG. 21B)body weight after 2-PMPA; (FIG. 21C) colon weight changes; and (FIG.21D) prolapse retraction after treatment. IL-10 KO mice (C57/B6; 3 monthold) were treated with 2-PMPA (100 mg/kg) for 2 weeks.*P<0.05;

FIG. 22 shows a flow-chart of an experimental design to address whetherthe PSMA inhibitor directly targets on colonic epithelial cells (CECs);CACO-2 cell lines or CECs isolated from WT and IL-10KO mice will beused;

FIG. 23A and FIG. 23B show the expression of CD103 on human intestinalDC: (FIG. 23A) FACS dot plot demonstrating identification of humancolonic CD103+DC from biopsies and surgical resection tissue obtainedfrom healthy controls or patients with active CD/UC (inflamed areas),via gating on viable cells according to forward and side scatter (notshown), HLA-DR versus lineage cocktail (CD3/CD14/CD16/CD19/CD34), andsubsequent CD103 histogram; and (FIG. 23B) summary graph representingall experiments (control: n=14; IBD: n=8). T-test was applied***p<0.001;

FIG. 24A, FIG. 24B, FIG. 24C and FIG. 24D show the expression of a4137on murine intestinal DC: (FIG. 24A) FACS dot plot demonstratingidentification of murine colonic CD103+DC as CD11c+MHC Class II+following gating on CD45+ live cells; (FIG. 24B) FACS dot plotdemonstrating α₄β₇ co-expression with CD103 (markers were co-expressedin all experiments). Histogram was gated on CD45+ live cells, andsubsequently MHC Class II+CD11c+ cells; (FIG. 24C) FACS histogramdemonstrating example of a4137 expression on murine colonic DC.Histogram was gated on CD45+ live cells, and subsequently MHC ClassII+CD11c+ cells; and (FIG. 24D) summary graph representing allexperiments (n=3 for both). T-test was applied: *p<0.05;

FIG. 25 shows PK results with compound JAM0388 after dosing animals at30 mg/kg equivalent of 2-PMPA by oral gavage and collecting brain andplasma samples at 30 min and 5 h. Brain and plasma samples werequantified for 2-PMPA using our previously published method (Rais et al,J Pharm Biomed Anal. 2014 January; 88:162-9). JAM0338 demonstratedexcellent release of 2-PMPA in plasma and levels were quantifiable evenat 5 h, showing sustained release of 2-PMPA from the prodrug. Brainlevels were low at both time points;

FIG. 26 is a schematic illustration demonstrating that GCPII cleavesNAAG to NAA and glutamate in the brain;

FIG. 27 demonstrates that GCPII inhibition increases NAAG in the brainby >30 fold measured by microdialysis;

FIG. 28A, FIG. 28B and FIG. 28C demonstrate that 2-PMPA elevates NAAGand improves cognitive function in EAE mice. FIG. 28A demonstrates thatmice show equal cognitive ability in Barnes maze paths on Day 1. FIG.28B shows that on Day 4), EAE mice treated with vehicle show deficit onlearning, while EAE mice treated with 2-PMPA have equal cognitivefunction to healthy control mice. FIG. 28C demonstrates that [NAAG] iselevated in EAE mice treated with 2PMPA; and

FIG. 29 shows plasma concentration time profiles of 2-PMPA followingi.p. (black line) and oral (red line) administration at 100 mg/kg inmice.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

I. Prodrugs of 2-PMPA

2-PMPA is a highly polar compound with multiple carboxylates and a zincbinding phosphonate group and it has negligible oral availability. Theusual way of solving this problem is by converting polar groups intoless polar functional derivatives. However, typical prodrug approachesincluding simple alkyl esters of the acids such as methyl, ethyl, andpropyl were attempted and were not successful, due to excess stabilityof these moieties on the carboxylates. Pivaloyloxymethyl (POM) andpropyloxycarbonyloxymethyl (POC) on phosphonate groups demonstrated theright combination of lability in vitro and provided the highest levelsof prodrug derived species when dosed orally. Even a compound with afree γ carboxylate demonstrated good bioavailability. However, none ofthese compounds released 2-PMPA in vivo to any appreciable extent. Thepresently disclosed subject matter shows that POM and POC on the bisphosphonate and the alpha carboxylate were ideal for enhancing thepermeability (approximately 20 fold), as well as release of the parentcompound upon oral dosing.

Structures of representative structures of 2-PMPA prodrugs are providedin Table 1. More particularly, the presently disclosed subject matterincludes the capping of the acidic functional groups of 2-PMPA. In someembodiments, the carboxylic acid groups of 2-PMPA were protected withalkyl esters. See for example, compounds 1, 2, and 3 of Table 1. Thesecarboxylic esters, however, unexpectedly were too stable in vivo to beeffective prodrugs. Protecting the phosphonate of 2-PMPA with forexample bis-POM (compound 4) or bis-POC (compound 5) while leaving thecarboxylates free, however, was not a feasible solution because of thechemical instability of such derivatives.

A combination of both approaches, i.e., protecting the carboxylic acidgroups with an alkyl ester and protecting the phosphonate with POM orPOC, e.g., compounds 6 and 7, provided compounds that exhibited goodpermeability. These compounds, however, were only converted to thecorresponding carboxylate ester, compound 1, which is stable in plasmaand did not exhibit the ability to release 2-PMPA.

Compounds including POM and POC on the bis-phosphonate of 2-PMPA and analkyl ester on the α-carboxylate, with a free γ-carboxylate, e.g.,compounds 8 and 9, exhibited good oral availability, but were onlyconverted to monoester 10.

The stability of the simple carboxylic ester, however, can be overcomeby introducing another POC or POM moiety on the α-carboxylate (compounds11 and 12, respectively). Such compounds exhibited sufficient chemicalstability, yet exhibited the potential to release 2-PMPA.

TABLE 1 Structures of Representative 2-PMPA Prodrugs and MetabolicProducts IOCB No./ Compound No. Structure MW 2-PMPA

226.12  1 TT-140113 JHU 2106

254.17  2 MK-797 JHU 2236

282.23  3 MK-801 JHU 2263

310.28  4

458.35  5

454.41  6 TT-010213 JHU 2110

486.41  7 MK-793 JHU 2234

472.38  8 TT-150313 JHU 2201

468.43  9

240.15 10 JAM0186 2-PMPATRIS-POC

574.47 11

582.57 12 TT-041212 JHU 2107

791.13 13 TT-250113 JHU 2108

838.90 14 TT-201212A JHU 2109

482.46 15 TT-010213 JHU 2110

486.40 16 TT-100113 JHU 2111

606.58 17 TT-280113 JHU 2112

604.60 18 MK798 JHU 2237

510.51 19 MK804 JHU 2264

482.46 20 MK806 JHU 2265

486.40 21 MK-795 JHU 2235

592.57 22 MK-799 JHU 2238

520.68 23 JAM0168 2-PMPA TRIS POM

568.55 24 JAM0195 JHU 2609

616.55 25 JAM0191 JHU 2608

543.46 26 JAM0196 JHU 2610

571.51 27 LTP023

522.44 28 LTP120

670.60 29 LTP124

670.60 30 JAM0388H

494.43 31 JAM0341H

540.50 32 TT-120814

450.29 33 TT-200714

450.29 34 TT-270514

540.41 35 TT-011214

674.45 36 TT-110814A

562.37

In yet other embodiments, fine tuning of the hydrolysis rate can beevaluated by a combination of POC and methyl-substituted POC, asillustrated by the following compounds:

Further directions in 2-PMPA prodrugs include the following approach,including more easily hydrolysable phenyl esters; anhydrides, anddioxolone esters employing paraoxonase for bioconversion:

Additionally, the following dioxolone esters and anhydride prodrugs of2-PMPA are contemplated:

Further examples of alternative carboxy-esters prodrugs of 2-PMPA alsoinclude:

Accordingly, in some embodiments, the presently disclosed subject matterprovides a compound of formula (I) or formula (II):

-   -   wherein:    -   each R₁, R₂, R₃, and R₄ is independently selected from the group        consisting of H, alkyl, Ar, —(CR₅R₆)_(n)—Ar,        —(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,        —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,        —(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇,        —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇,        —(CR₅R₆)_(n)—NR₈R₉, and —(CR₅R₆)_(n)—C(═O)—NR₈R₉;    -   wherein:    -   n is an integer from 1 to 20;    -   m is an integer from 1 to 20;    -   each R_(3′) and R_(4′) are independently H or alkyl;    -   each R₅ and R₆ is independently selected from the group        consisting of H, alkyl, and alkylaryl;    -   each R₇ is independently straightchain or branched alkyl;    -   Ar is aryl, substituted aryl, heteroaryl or substituted        heteroaryl; and    -   R₈ and R₉ are each independently H or alkyl; and    -   pharmaceutically acceptable salts thereof.

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (I) is in

In particular embodiments, the compound of formula (I) is selected fromthe group consisting of:

In particular embodiments, the compound of formula (II) is

In particular embodiments, the compound of formula (II) is

II. Pharmaceutical Compositions and Administration

In another aspect, the present disclosure provides a pharmaceuticalcomposition including a compound of formula (I), or a compound offormula (II), alone or in combination with one or more additionaltherapeutic agents in admixture with a pharmaceutically acceptableexcipient. One of skill in the art will recognize that thepharmaceutical compositions include the pharmaceutically acceptablesalts of the compounds described above.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000).

The compounds according to the disclosure are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, andfrom 5 to 40 mg per day are examples of dosages that may be used. Anon-limiting dosage is 10 to 30 mg per day. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Pharmaceutically acceptable saltsinclude, for example, acetate, benzoate, bromide, carbonate, citrate,gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate,pamoate (embonate), phosphate, salicylate, succinate, sulfate, ortartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articullar, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions, such as in physiologically compatiblebuffers such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP:povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

III. Methods for Treating a Disease or Disorder

The presently disclosed compounds, which are orally bioavailableprodrugs of 2-PMPA, allow a clinically acceptable dosing paradigm fordiseases or conditions wherein excess PSMA/GCPII activity is implicated.These diseases or conditions include, but are not limited to,neurodegenerative disease such as amyotrophic lateral sclerosis (ALS),Parkinson's disease (PD), Alzheimer's disease (AD), Huntington'sdisease, dementia with Lewy Bodies (DLB), schizophrenia, pain, epilepsy,stroke, and traumatic brain injury (TBI), as well as multiple sclerosis(MS), cancer, angiogenesis and inflammatory bowel disease. As usedherein, a “neurodegenerative disease” is a disease or condition thatresults in the progressive loss of the structure and/or function ofneurons in a subject.

As used herein, the terms “PSMA” or “PSMA polypeptide” refer to anaturally occurring or endogenous PSMA and to proteins having an aminoacid sequence which is the same as that of a naturally occurring orendogenous PSMA (e.g., recombinant proteins). Accordingly, as definedherein, the term includes mature PSMA, glycosylated or unglycosylatedPSMA proteins, polymorphic or allelic variants, and other isoforms ofPSMA (e.g., produced by alternative splicing or other cellularprocesses).

As used herein, an “inhibitor” of PSMA is a molecule that decreases orinhibits the activity of PSMA when administered. The inhibitor mayinteract with PSMA directly or may interact with another molecule thatresults in a decrease in the activity of PSMA.

The presently disclosed subject matter shows that there is a markedelevation or excess of PSMA activity in subjects with certain diseasesor conditions. As used herein, the term “excess PSMA activity” means anincrease of PSMA activity in a subject with a disease or condition ascompared to the PSMA activity in a subject without a similar disease orcondition, such as an increase of approximately 100%, 200%, 300%, 400%,500%, 600%, 700%, 800%, 900%, 1000%, or more.

In some embodiments, the presently disclosed subject matter providesmethods for inhibiting the excess PSMA activity found in a subject witha disease or condition. As used herein, the term “inhibit” means todecrease or diminish the excess PSMA activity found in a subject. Theterm “inhibit” also may mean to decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease orcondition. Inhibition may occur, for e.g., by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% compared to anuntreated control subject or a subject without the disease or disorder.

In general, the presently disclosed methods result in a decrease in theseverity of a disease or condition in a subject. The term “decrease” ismeant to inhibit, suppress, attenuate, diminish, arrest, or stabilize asymptom of a disease or condition.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disease or condition, and/orsymptoms associated therewith. It will be appreciated that, although notprecluded, treating a disease or condition does not require that thedisorder, condition or symptoms associated therewith be completelyeliminated.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for treating a disease or a condition, the methodcomprising administering to a subject in need of treatment thereof, acompound of formula (I), a compound of formula (II), or a pharmaceuticalcomposition thereof, in an amount effective for treating the disease orcondition.

In particular embodiments, the disease or condition is selected from thegroup consisting of a neurodegenerative disease, multiple sclerosis(MS), cancer, angiogenesis, and inflammatory bowel disease.

In certain embodiments, the neurodegenerative disease is selected fromthe group consisting of amyotrophic lateral sclerosis (ALS), Parkinson'sdisease (PD), Alzheimer's disease (AD), Huntington's disease, dementiawith Lewy Bodies (DLB), schizophrenia, pain, epilepsy, stroke, andtraumatic brain injury (TBI).

In some embodiments, the disease or condition results in excess PSMAactivity. In such aspects, the method further comprises inhibiting theexcess PSMA activity when the compound of formula (I), the compound offormula (II), or a pharmaceutical composition thereof, is administered.

IV. Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

While the following terms in relation to compounds of formula (I) arebelieved to be well understood by one of ordinary skill in the art, thefollowing definitions are set forth to facilitate explanation of thepresently disclosed subject matter. These definitions are intended tosupplement and illustrate, not preclude, the definitions that would beapparent to one of ordinary skill in the art upon review of the presentdisclosure.

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability, as appreciated byone skilled in this art, to change one functional group for anotherfunctional group provided that the valency of all atoms is maintained.When more than one position in any given structure may be substitutedwith more than one substituent selected from a specified group, thesubstituent may be either the same or different at every position. Thesubstituents also may be further substituted (e.g., an aryl groupsubstituent may have another substituent off it, such as another arylgroup, which is further substituted, for example, with fluorine at oneor more positions).

Where substituent groups or linking groups are specified by theirconventional chemical formulae, written from left to right, they equallyencompass the chemically identical substituents that would result fromwriting the structure from right to left, e.g., —CH₂O— is equivalent to—OCH₂—; —C(═O)O— is equivalent to —OC(═O)—; —OC(═O)NR— is equivalent to—NRC(═O)O—, and the like.

When the term “independently selected” is used, the substituents beingreferred to (e.g., R groups, such as groups R₁, R₂, and the like, orvariables, such as “m” and “n”), can be identical or different. Forexample, both R₁ and R₂ can be substituted alkyls, or R₁ can be hydrogenand R₂ can be a substituted alkyl, and the like.

The terms “a,” “an,” or “a(n),” when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

A named “R” or group will generally have the structure that isrecognized in the art as corresponding to a group having that name,unless specified otherwise herein. For the purposes of illustration,certain representative “R” groups as set forth above are defined below.

Description of compounds of the present disclosure are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The term hydrocarbon, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. As would be known to one skilled in this art, allvalencies must be satisfied in making any substitutions. The hydrocarbonmay be unsaturated, saturated, branched, unbranched, cyclic, polycyclic,or heterocyclic. Illustrative hydrocarbons are further defined hereinbelow and include, for example, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl,methoxy, diethylamino, and the like.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, acyclic or cyclic hydrocarbon group, or combination thereof,which may be fully saturated, mono- or polyunsaturated and can includedi- and multivalent groups, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). In particular embodiments, theterm “alkyl” refers to C₁₋₂₀ inclusive, linear (i.e., “straight-chain”),branched, or cyclic, saturated or at least partially and in some casesfully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon radicalsderived from a hydrocarbon moiety containing between one and twentycarbon atoms by removal of a single hydrogen atom.

Representative saturated hydrocarbon groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, sec-pentyl, iso-pentyl, neopentyl, n-hexyl,sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.

“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁₋₈straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls. Alkyl groups can optionallybe substituted (a “substituted alkyl”) with one or more alkyl groupsubstituents, which can be the same or different. The term “alkyl groupsubstituent” includes but is not limited to alkyl, substituted alkyl,halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio,aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl.There can be optionally inserted along the alkyl chain one or moreoxygen, sulfur or substituted or unsubstituted nitrogen atoms, whereinthe nitrogen substituent is hydrogen, lower alkyl (also referred toherein as “alkylaminoalkyl”), or aryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂₅—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to twoor three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

As described above, heteroalkyl groups, as used herein, include thosegroups that are attached to the remainder of the molecule through aheteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR, and/or —SO₂R′.Where “heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl,aryl, or substituted aryl, thus providing a heterocyclic group.Representative monocyclic cycloalkyl rings include cyclopentyl,cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings includeadamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl, and fused ring systems, such as dihydro- andtetrahydronaphthalene, and the like.

The term “cycloalkylalkyl,” as used herein, refers to a cycloalkyl groupas defined hereinabove, which is attached to the parent molecular moietythrough an alkyl group, also as defined above. Examples ofcycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

The terms “cycloheteroalkyl” or “heterocycloalkyl” refer to anon-aromatic ring system, unsaturated or partially unsaturated ringsystem, such as a 3- to 10-member substituted or unsubstitutedcycloalkyl ring system, including one or more heteroatoms, which can bethe same or different, and are selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si),and optionally can include one or more double bonds.

The cycloheteroalkyl ring can be optionally fused to or otherwiseattached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbonrings. Heterocyclic rings include those having from one to threeheteroatoms independently selected from oxygen, sulfur, and nitrogen, inwhich the nitrogen and sulfur heteroatoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. In certainembodiments, the term heterocylic refers to a non-aromatic 5-, 6-, or7-membered ring or a polycyclic group wherein at least one ring atom isa heteroatom selected from O, S, and N (wherein the nitrogen and sulfurheteroatoms may be optionally oxidized), including, but not limited to,a bi- or tri-cyclic group, comprising fused six-membered rings havingbetween one and three heteroatoms independently selected from theoxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfurheteroatoms may be optionally oxidized, (iii) the nitrogen heteroatommay optionally be quaternized, and (iv) any of the above heterocyclicrings may be fused to an aryl or heteroaryl ring. Representativecycloheteroalkyl ring systems include, but are not limited topyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl,morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and thelike.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

An unsaturated alkyl group is one having one or more double bonds ortriple bonds. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl.”

More particularly, the term “alkenyl” as used herein refers to amonovalent group derived from a C₁₋₂₀ inclusive straight or branchedhydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. Alkenyl groups include, for example,ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-buten-1-yl,pentenyl, hexenyl, octenyl, and butadienyl.

The term “cycloalkenyl” as used herein refers to a cyclic hydrocarboncontaining at least one carbon-carbon double bond. Examples ofcycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “alkynyl” as used herein refers to a monovalent group derivedfrom a straight or branched C₁₋₂₀ hydrocarbon of a designed number ofcarbon atoms containing at least one carbon-carbon triple bond. Examplesof “alkynyl” include ethynyl, 2-propynyl (propargyl), 1-propynyl,pentynyl, hexynyl, heptynyl, and allenyl groups, and the like.

The term “alkylene” by itself or a part of another substituent refers toa straight or branched bivalent aliphatic hydrocarbon group derived froman alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—,—CH₂CsCCH₂—, —CH₂CH₂CH(CH₂CH₂CH₃)CH₂—, —(CH₂)_(q)—N(R)—(CH₂)_(r)—,wherein each of q and r is independently an integer from 0 to about 20,e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl(—O—CH₂—O—); and ethylenedioxyl (—O—(CH₂)₂—O—). An alkylene group canhave about 2 to about 3 carbon atoms and can further have 6-20 carbons.Typically, an alkyl (or alkylene) group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being someembodiments of the present disclosure. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent group derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)OR′— represents both —C(O)OR′—and —R′OC(O)—.

The term “aryl” means, unless otherwise stated, an aromatic hydrocarbonsubstituent that can be a single ring or multiple rings (such as from 1to 3 rings), which are fused together or linked covalently. The term“heteroaryl” refers to aryl groups (or rings) that contain from one tofour heteroatoms (in each separate ring in the case of multiple rings)selected from N, O, and S, wherein the nitrogen and sulfur atoms areoptionally oxidized, and the nitrogen atom(s) are optionallyquaternized. A heteroaryl group can be attached to the remainder of themolecule through a carbon or heteroatom. Non-limiting examples of aryland heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent forms of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the terms “arylalkyl” and“heteroarylalkyl” are meant to include those groups in which an aryl orheteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl, furylmethyl, and the like) including those alkyl groupsin which a carbon atom (e.g., a methylene group) has been replaced by,for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxy)propyl, and the like). However, the term “haloaryl,” asused herein is meant to cover only aryls substituted with one or morehalogens.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g. “3 to 7 membered”), the term “member” refers toa carbon or heteroatom.

Further, a structure represented generally by the formula:

as used herein refers to a ring structure, for example, but not limitedto a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a 7-carbon, and thelike, aliphatic and/or aromatic cyclic compound, including a saturatedring structure, a partially saturated ring structure, and an unsaturatedring structure, comprising a substituent R group, wherein the R groupcan be present or absent, and when present, one or more R groups caneach be substituted on one or more available carbon atoms of the ringstructure. The presence or absence of the R group and number of R groupsis determined by the value of the variable “n,” which is an integergenerally having a value ranging from 0 to the number of carbon atoms onthe ring available for substitution. Each R group, if more than one, issubstituted on an available carbon of the ring structure rather than onanother R group. For example, the structure above where n is 0 to 2would comprise compound groups including, but not limited to:

and the like.

A dashed line representing a bond in a cyclic ring structure indicatesthat the bond can be either present or absent in the ring. That is, adashed line representing a bond in a cyclic ring structure indicatesthat the ring structure is selected from the group consisting of asaturated ring structure, a partially saturated ring structure, and anunsaturated ring structure.

The symbol (‘

’) denotes the point of attachment of a moiety to the remainder of themolecule.

When a named atom of an aromatic ring or a heterocyclic aromatic ring isdefined as being “absent,” the named atom is replaced by a direct bond.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl,” “phosphonate,” and “sulfonate”as well as their divalent derivatives) are meant to include bothsubstituted and unsubstituted forms of the indicated group. Optionalsubstituents for each type of group are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative groups (including those groups oftenreferred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —C(O)NR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such groups. R′, R″, R′″ and R″″ each mayindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. As used herein, an“alkoxy” group is an alkyl attached to the remainder of the moleculethrough a divalent oxygen. When a compound of the disclosure includesmore than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant toinclude, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. Fromthe above discussion of substituents, one of skill in the art willunderstand that the term “alkyl” is meant to include groups includingcarbon atoms bound to groups other than hydrogen groups, such ashaloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃,—C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl groups above, exemplarysubstituents for aryl and heteroaryl groups (as well as their divalentderivatives) are varied and are selected from, for example: halogen,—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′,—C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)OR′,—NR—C(NR′R″R′″)═NR′″′, —NR—C(NR′R″)═NR′″—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxo, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on aromatic ring system; and where R′, R″, R′″ and R′″′may be independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl. When a compound of the disclosure includesmore than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″ groups when morethan one of these groups is present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR′— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4.

One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where sand d are independently integers of from 0 to 3, and X′ is —O—, —NR′—,—S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″ and R′″may be independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

As used herein, the term “acyl” refers to an organic acid group whereinthe —OH of the carboxyl group has been replaced with another substituentand has the general formula RC(═O)—, wherein R is an alkyl, alkenyl,alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic groupas defined herein). As such, the term “acyl” specifically includesarylacyl groups, such as an acetylfuran and a phenacyl group. Specificexamples of acyl groups include acetyl and benzoyl.

The terms “alkoxyl” or “alkoxy” are used interchangeably herein andrefer to a saturated (i.e., alkyl-O—) or unsaturated (i.e., alkenyl-O—and alkynyl-O—) group attached to the parent molecular moiety through anoxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are aspreviously described and can include C₁₋₂₀ inclusive, linear, branched,or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including,for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl,sec-butoxyl, t-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and thelike.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, a methoxyethyl or an ethoxymethyl group.

“Aryloxyl” refers to an aryl-O— group wherein the aryl group is aspreviously described, including a substituted aryl. The term “aryloxyl”as used herein can refer to phenyloxyl or hexyloxyl, and alkyl,substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl.

“Alkoxycarbonyl” refers to an alkyl-O—CO— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and t-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—CO— group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—CO— group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an amide group of the formula —CONH₂.“Alkylcarbamoyl” refers to a R′RN—CO— group wherein one of R and R′ ishydrogen and the other of R and R′ is alkyl and/or substituted alkyl aspreviously described. “Dialkylcarbamoyl” refers to a R′RN—CO— groupwherein each of R and R′ is independently alkyl and/or substituted alkylas previously described.

The term carbonyldioxyl, as used herein, refers to a carbonate group ofthe formula —O—CO—OR.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

The term “amino” refers to the —NH₂ group and also refers to a nitrogencontaining group as is known in the art derived from ammonia by thereplacement of one or more hydrogen radicals by organic radicals. Forexample, the terms “acylamino” and “alkylamino” refer to specificN-substituted organic radicals with acyl and alkyl substituent groupsrespectively.

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. More particularly, the terms alkylamino,dialkylamino, and trialkylamino as used herein refer to one, two, orthree, respectively, alkyl groups, as previously defined, attached tothe parent molecular moiety through a nitrogen atom. The term alkylaminorefers to a group having the structure —NHR′ wherein R′ is an alkylgroup, as previously defined; whereas the term dialkylamino refers to agroup having the structure —NR′R″, wherein R′ and R″ are eachindependently selected from the group consisting of alkyl groups. Theterm trialkylamino refers to a group having the structure —NR′R″R′″,wherein R′, R″, and R′″ are each independently selected from the groupconsisting of alkyl groups. Additionally, R′, R″, and/or R′″ takentogether may optionally be —(CH₂)_(k)— where k is an integer from 2 to6. Examples include, but are not limited to, methylamino, dimethylamino,ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino,iso-propylamino, piperidino, trimethylamino, and propylamino.

The amino group is —NR′R″, wherein R′ and R″ are typically selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

The terms alkylthioether and thioalkoxyl refer to a saturated (i.e.,alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groupattached to the parent molecular moiety through a sulfur atom. Examplesof thioalkoxyl moieties include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed. “Aroylamino” refers to an aroyl-NH— group wherein aroyl is aspreviously described.

The term “carbonyl” refers to the —(C═O)— group.

The term “carboxyl” refers to the —COOH group. Such groups also arereferred to herein as a “carboxylic acid” moiety.

The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,”are meant to include monohaloalkyl and polyhaloalkyl. For example, theterm “halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “oxo” as used herein means an oxygen atom that is double bondedto a carbon atom or to another element.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄ group.

The term thiohydroxyl or thiol, as used herein, refers to —SH.

The term ureido refers to a urea group of the formula —NH—CO—NH₂.

Unless otherwise explicitly defined, a “substituent group,” as usedherein, includes a functional group selected from one or more of thefollowing moieties, which are defined herein:

(A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, substituted with at least one substituent selected from oxo,—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described hereinabove fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

Throughout the specification and claims, a given chemical formula orname shall encompass all tautomers, congeners, and optical- andstereoisomers, as well as racemic mixtures where such isomers andmixtures exist.

Certain compounds of the present disclosure possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present disclosure. The compounds ofthe present disclosure do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present disclosure ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefenic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure. The term“tautomer,” as used herein, refers to one of two or more structuralisomers which exist in equilibrium and which are readily converted fromone isomeric form to another.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C— or ¹⁴C-enriched carbonare within the scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present disclosure, whether radioactive or not, are encompassedwithin the scope of the present disclosure.

As used herein the term “monomer” refers to a molecule that can undergopolymerization, thereby contributing constitutional units to theessential structure of a macromolecule or polymer.

A “polymer” is a molecule of high relative molecule mass, the structureof which essentially comprises the multiple repetition of unit derivedfrom molecules of low relative molecular mass, i.e., a monomer.

As used herein, an “oligomer” includes a few monomer units, for example,in contrast to a polymer that potentially can comprise an unlimitednumber of monomers. Dimers, trimers, and tetramers are non-limitingexamples of oligomers.

The compounds of the present disclosure may exist as salts. The presentdisclosure includes such salts. Examples of applicable salt formsinclude hydrochlorides, hydrobromides, sulfates, methanesulfonates,nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g.(+)-tartrates, (−)-tartrates or mixtures thereof including racemicmixtures, succinates, benzoates and salts with amino acids such asglutamic acid. These salts may be prepared by methods known to thoseskilled in art. Also included are base addition salts such as sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present disclosure containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acceptable acid addition salts include those derived frominorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the present disclosure contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents.

Certain compounds of the present disclosure can exist in unsolvatedforms as well as solvated forms, including hydrated forms. In general,the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present disclosure. Certaincompounds of the present disclosure may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for theuses contemplated by the present disclosure and are intended to bewithin the scope of the present disclosure.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present disclosurecontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentdisclosure contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike {see, for example, Berge et al, “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present disclosure contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts.

In addition to salt forms, the present disclosure provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentdisclosure. Additionally, prodrugs can be converted to the compounds ofthe present disclosure by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present disclosure when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in T. W.Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd ed.John Wiley & Sons (1999). It may be advantageous, where differentprotecting groups are employed, that each (different) protective groupbe removable by a different means. Protective groups that are cleavedunder totally disparate reaction conditions allow differential removalof such protecting groups. For example, protective groups can be removedby acid, base, and hydrogenolysis. Groups such as trityl,dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile andmay be used to protect carboxy and hydroxy reactive moieties in thepresence of amino groups protected with Cbz groups, which are removableby hydrogenolysis, and Fmoc groups, which are base labile. Carboxylicacid and hydroxy reactive moieties may be blocked with base labilegroups such as, without limitation, methyl, ethyl, and acetyl in thepresence of amines blocked with acid labile groups such as tert-butylcarbamate or with carbamates that are both acid and base stable buthydrolytically removable.

Carboxylic acid and hydroxy reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups capable of hydrogen bonding with acids may be blockedwith base labile groups such as Fmoc. Carboxylic acid reactive moietiesmay be blocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups may be blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(O)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

Typical blocking/protecting groups include, but are not limited to thefollowing moieties:

The subject treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease. Thus,the terms “subject” and “patient” are used interchangeably herein.

In general, the “effective amount” of an active agent or drug deliverydevice refers to the amount necessary to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of an agent or device may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the composition of the encapsulating matrix, the target tissue, and thelike.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Methods

In Vitro Stability Studies: The stock solution for most prodrugs wasprepared as a 10 mM solution in DMSO except JHU 2107, which wassolubilized in THF (tetrahydrofuran) to carry out the in vitro studies.

The chemical stability of prodrugs was evaluated using simulated gastricfluid (pH 1.2) and Hanks' Balanced Salt Solution (HBSS) buffer (pH 7.4).Briefly, prodrugs were spiked (10 μM) in respective solutions andincubated at 37° C. for 1 h. At predetermined time points (0, 30 and 60min), aliquots of 100 μL were removed and diluted with 1041 of water.Prodrug disappearance was monitored using the developed liquidchromatography and tandem mass spectrometry (LC/MS/MS) method describedbelow.

For metabolic stability, plasma (mouse, dog, monkey and human) and livermicrosomes (mouse, dog, monkey and human) were used. For stability,prodrugs (10 μM) were spiked in each matrix and incubated in an orbitalshaker at 37° C. At predetermined times (0, 30 and 60 min), 100 μLaliquots of the mixture in triplicate were removed and the reactionquenched by addition of three times the volume of ice cold acetonitrilespiked with the internal standard (losartan 5 μM). The samples werevortexed for 30 s and centrifuged 12000 g for 10 min. 50 μL supernatantdiluted with 50 μL water was transferred to a 250 μL polypropylene vialsealed with a Teflon cap. Prodrug disappearance was monitored over timeusing a liquid chromatography and tandem mass spectrometry (LC/MS/MS)method as described below.

For LC/MS/MS, prodrugs were separated with Thermo Scientific Accela UPLCsystem coupled to Accela open autosampler on an Agilent C18 (100×2.1 mmid) UPLC column. The autosampler was temperature controlled andoperating at 10° C. The mobile phase used for the chromatographicseparation was composed of acetonitrile/water containing 0.1% formicacid and was run at a flow rate of 0.5 mL/minute for 4.5 minutes usinggradient elution. The column effluent was monitored using TSQ Vantagetriple-quadrupole mass spectrometric detector, equipped with anelectrospray probe set in the positive ionization mode. Samples wereintroduced into the ionization source through a heated nebulized probe(350° C.).

For quantification of compound remaining, disappearance of prodrugs wasmeasured from ratio of peak areas of analyte to IS. Percentage remainingwas calculated in the following manner:

$\frac{{{Avg}.{Response}}*{@60}\mspace{14mu}\min}{{{Avg}.{{Response}\mspace{11mu}@0}}\mspace{14mu}\min} \times 100$

-   where response=[(Area of analyte)/(Area of internal standard)]-   Average response is average of two samples at each time point.

In Vivo Pharmacokinetics of 2-PMPA Prodrugs in Rodent (Mice) andNon-Rodent (Dogs) Species: Prodrugs were dosed peroral (30 mg/kg equiv.2-PMPA) in mice at a dosing volume of 1 mL/kg. Blood was obtained viacardiac puncture and tissue dissected at 0 min, 15 min, 30 min, 1 h, 2h, and 4 h post dose (n=3 per time point). Single time point studieswere conducted at 30 min (N=3) following dosing. Plasma was harvestedfrom blood by centrifugation. Mean concentration-time data was used forpharmacokinetic (PK) analysis. Non-compartmental-analysis module inWinNonlin® (version 5.3) was used to assess pharmacokinetic parameters.Peak plasma concentrations (C_(max)) and time to C_(max) (T_(max)) werethe observed values. Area under the curve (AUC) was calculated bylog-linear (p.o.) trapezoidal rule to the end of sample collection(AUClast) and extrapolated to infinity (AUC_(0-∞)) by dividing the lastquantifiable concentration by the terminal disposition rate constant(ke). Terminal half-life (t_(1/2)) was estimated from first orderkinetics: t_(1/2)=0.693/k_(e). The goal was to find prodrugs yieldingoral bioavailability % F≥30%.

For pharmacokinetics in beagle dogs, animals were dosed with 2-PMPAprodrug (10 mg/kg equivalent 2-PMPA) p.o. (by mouth). Blood samples werecollected from the jugular vein (˜1 mL) via direct venipuncture, placedinto potassium oxalate with sodium fluoride tubes, and maintained on wetice until processed. Blood samples were centrifuged at a temperature of4° C., at 3000×g, for 5 minutes. Blood samples were maintained chilledthroughout processing. Plasma was collected in tubes and flash frozen.Samples were stored in a freezer set to maintain −60° C. to −80° C.until further analysis.

Bioanalysis of 2-PMPA Prodrugs in Plasma and Tissue: 2-PMPAconcentrations in plasma and tissue samples were determined using twodifferent methods (FIG. 2). Method 1 showed the total amount of theprodrug in the sample following oral dosing (a measure of thedisappearance of the prodrug) and method 2 evaluated the specificrelease of 2-PMPA in plasma and tissues from the prodrug. In vitroscreening showed metabolic instability of almost all the prodrugstested.

Method 1 involved use of a strong derivatizing reagent, n-butanol with3N HCl, which converted the prodrug and its metabolites including 2-PMPAinto a 2-PMPA butyl ester to obtain the total exposures from theprodrug. Briefly, prior to extraction, frozen samples were thawed onice. For plasma extraction, 50 μL of the calibration standards orsamples were transferred into silanized microcentrifuge tubes. Samplepreparation involved a single liquid extraction by addition of 300 μL ofmethanol as extraction solution with internal standard (i.e., 5 μM of2-PMSA in methanol), followed by vortexing for 30 s and thencentrifugation at 12000 g for 10 min. Supernatant was transferred (˜250μL) and evaporated to dryness at 40° C. under a gentle stream ofnitrogen. The residue was reconstituted with 100 μL of derivatizingagent, n-butanol with 3N HCl, and samples were vortexed. The sampleswere heated at ˜60° C. in a shaking water bath for 30 min. At the end of30 min, the derivatized samples were allowed to cool at room temperatureand dried again for removal of derivatizing reagent, under a gentlestream of nitrogen. The residue was reconstituted in 100 μL of 30%acetonitrile in water v/v. The samples were vortexed and centrifugedagain. ˜80 μL supernatant was transferred to a 250 μL, polypropylenevial sealed with a Teflon cap and a volume of 10 μL was injected ontothe ultra-performance liquid chromatography (UPLC) instrument forquantitative analysis. Chromatographic analysis was performed using anAccela™ ultra high-performance system consisting of an analytical pump,and an autosampler coupled with TSQ Vantage mass spectrometer (ThermoFisher Scientific Inc., Waltham Mass.). Separation of the analyte frompotentially interfering material was achieved at ambient temperatureusing Agilent Eclipse Plus column (100×2.1 mm i.d.) packed with a 1.8 μmC18 stationary phase. The mobile phase used was composed of 0.1% formicacid in acetonitrile and 0.1% formic acid in H₂O with gradient elution,starting with 20% (organic) linearly increasing to 65% up to 2.5 min,maintaining at 65% (2.5-3.5 min) and reequilibrating to 30% by 5 min.The total run time for each analyte was 5.0 min. The [M+H]⁺ iontransitions of derivatized 2-PMPA at m/z 325.522>121.296, 195.345 andthat of the internal standard at m/z 339.537>191.354, 149.308, weremonitored.

Method 2 was a gentle method to evaluate specific release of 2-PMPA inplasma and tissues from prodrug. Briefly, 2-PMPA was extracted fromplasma by protein precipitation with 5× methanol containing2-(phosphonomethyl) succinic acid (2-PMSA; 1 μM) as an internalstandard. For brain tissue extraction, the samples were weighed in a 1.7mL silanized tubes to which 4 times the volume of methanol (dilution1:5) was added. The tissues were stored in −20° C. for 1 h and thenhomogenized. The calibration curve for the tissues was developed usingnaïve mouse brains from untreated animals as a matrix. For sciaticnerve, the nerves were weighed and homogenized in 504 methanol and thecalibration curve was developed using naïve sciatic nerves fromuntreated animals as a matrix. The samples were vortexed andcentrifuged. For tissue extraction, either 50 μL (brain) or 25 μL(sciatic nerve) of the calibration standards or samples were transferredinto silanized microcentrifuge tubes. Sample preparation involved asingle liquid extraction by addition of 150 μL of methanol as extractionsolution with internal standard (i.e., 5 μM of 2-PMSA in methanol).Supernatant was dried under a gentle stream of nitrogen at 45° C. andthe residue reconstituted with 75 μL of acetonitrile and vortexed. 25 μLof derivatizing agentN-tert-Butyldimethysilyl-N-methyltrifluoro-acetamide (MTBSTFA) was addedto microcentrifuge tubes, vortexed, and heated at ˜60° C. for 40 min. Atthe end of 40 min, the derivatized samples ˜75 μL were transferred to a250 μL polypropylene vials and were analyzed via LC/MS/MS.Chromatographic analysis was performed using an Accela™ ultrahigh-performance system consisting of an analytical pump, and anautosampler coupled with TSQ Vantage mass spectrometer (Thermo FisherScientific Inc., Waltham Mass.). Separation of the analyte frompotentially interfering material was achieved at ambient temperatureusing Waters X-Terr®, RP18, 3.5 μm, and (2.1×50 mm). The mobile phaseused was composed of 0.1% formic acid in acetonitrile and 0.1% formicacid in H₂O with gradient elution, starting with 90% (organic) linearlyincreasing to 99% up to 2.5 min, maintaining at 99% (2.5-4.0 min) andreequilibrating to 90% by 5 min. The total run time for each analyte was5.0 min. Chromatographic analysis will be performed on Accela UPLC. The[M+H]⁺ ion transitions of derivatized 2-PMPA at m/z 683.0>551.4 and thatof the internal standard at m/z 669.0>537.2 were monitored with thetotal run time of 5 min.

$\frac{{{Avg}.{Response}}*{@60}\mspace{14mu}\min}{{{Avg}.{{Response}\mspace{11mu}@0}}\mspace{14mu}\min} \times 100$

-   Where, Response=[(Area of analyte)/(Area of internal standard)]-   Average response is average of two samples at each time point.

Example 2 Compound Preparation

General Procedures: The ¹H NMR spectra were measured at 400.13. ¹H NMRspectra are standardized to the internal signal of TMS (δ 0.0, CDCl₃).The chemical shifts are given in 8-scale, the coupling constants J aregiven in Hz. The IR spectra were measured in CHCl₃ on FT-IR spectrometerBruker Equinox 55. Low and high resolution CI mass spectra were measuredusing an orthogonal acceleration time-of-flight (OA-TOF) massspectrometer (GCT premier, Waters) at an ionising voltage of 70 eV, them/z values are given with their relative intensities (%). The spectrawere recorded in positive mode and the source temperature was 150° C.Methane was present as a reagent gas in the CI source. For exactmeasurement the spectra were internally calibrated using Heptacosa or2,4,6-tris(trifluoromethyl)-1,3,5-triazine (Metri). The ESI mass spectrawere recorded with a ZQ micromass mass spectrometer (Waters) equippedwith an ESCi multi-mode ion source and controlled by MassLynx software.THF was freshly distilled from sodium/benzophenone under nitrogen. Theflash chromatography was performed on Silica gel 60 (0.040-0.063 mm,Fluka).

JAM0105

This compound was prepared from known literature. ¹H NMR and ¹³C NMRspectra were in agreement with the published data.

JAM0106

The same method of preparation as for previous compound JAM0131.Compound JAM0105 (6.62 g, 21.62 mmol), N,N-Dimethylmethyleneiminiumiodide (10 g, 54.05 mmol, 2.5 equiv.) Absolute methanol (265 mL).Reaction mixture was stirred at 65° C. h. The organic solvent wasevaporated in vacuo. The residue was filtered through pad of silica gel(hexane-ethyl acetate 5:1) to afford the desired product (5.02 g, 94%)as an oil. 1H NMR and 13C NMR spectra were in agreement with thepublished data.

JAM0113

The same method of preparation as for previous compound JAM0106.Compound JAM0105 (6.62 g, 21.62 mmol), N,N-Dimethylmethyleneiminiumiodide (10 g, 54.05 mmol, 2.5 equiv.) Absolute ethanol (265 mL).Reaction mixture was stirred at 78° C. overnight. The organic solventwas evaporated in vacuo. The residue was filtered through pad of silicagel (hexane-ethyl acetate 10:1 to 5:1) to afford the desired product(5.25 g, 93%) as an oil. 1H NMR and 13C NMR spectra were in agreementwith the published data.

JAM0131

A dry Schlenk flask was charged with the previous compound JAM0105 (6.62g, 21.62 mmol), N,N-Dimethylmethyleneiminium iodide (10 g, 54.05 mmol,2.5 equiv.) and then it was flushed with argon. Absolute t-BuOH (265 mL)was added to the flask and the mixture was stirred at 65° C. for 48 h.The organic solvent was evaporated in vacuo. The residue was filteredthrough pad of silica gel (hexane-ethyl acetate 5:1) to afford thedesired product (5 g, 80%) as an oil.

ESI MS: 313 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₂₂O₄Na 313.14103; found 313.14106.

JAM0109

The same method of preparation as for previous compound JAM0149. Diethylphosphite (2.6 mL, 20.14 mmol), A solution of trimethylaluminium (2 M inhexanes, 10 mL, 20.14 mmol, 1.0 equiv.) JAM0106 (5 g, 20.14 mmol, 1.0equiv.) dichloromethane (70 mL). Filtration through pad of silica gel(hexane-ethyl acetate 1:1 to 3:1) Product (7.3 g, 94%) as an oil.

ESI MS: 323 (M+Na⁺). HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found340.22805.

JAM0114

The same method of preparation as for previous compound JAM0149. Diethylphosphite (2.58 mL, 20 mmol), A solution of trimethylaluminium (2 M inhexanes, 10 mL, 20 mmol, 1.0 equiv.) JAM0113 (5.25 g, 20 mmol, 1.0equiv.) dichloromethane (70 mL). Filtration through pad of silica gel(hexane-ethyl acetate 1:1 to 3:1) Product (7.5 g, 94%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0149

Diethyl phosphite (8.5 mL, 66.1 mmol, 1 equiv.) was dissolved inabsolute dichloromethane (57 mL) under argon and cooled to 0° C. Asolution of trimethyl aluminium (2 M in hexanes, 33 mL, 66.1 mmol, 1equiv.) was added dropwise and the solution was stirred at 0° C. for 30min. Solution of the compound JAM0131 (19.2 g, 66.1 mmol, 1 equiv) indichloromethane (171 mL) was added and the cooling bath was removed. Thereaction mixture was then stirred at room temperature overnight. Thereaction was quenched with 2 N hydrochloric acid (40 mL). Then it wasextracted with diethyl ether (3×40 mL), the combined organic layers werewashed with water (40 mL), brine (40 mL), and dried over anhydrousMgSO4. The evaporation of the solvents afforded an oil, which wasfiltered through pad of silica gel (hexane-ethyl acetate 3:1 to 1:1) toafford the desired product (28.3 g, 94%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0112

A Schlank flask was charged with Ethyl acrylate (15 mL, 0.14 mol) underargon, then TDAP (5 mL, 28 mmol, 20 mol %) was slowly added and thereactin mixture was stirred at 60° C. for 2 h. Product was destillatedof on Kugelrohr aparatus (100° C. at 0.1 mbar). 1H NMR and 13C NMRspectra were in agreement with the published data.

JAM0115

A Schlank flask was charged with ethyl acrylate (15 mL, 0.12 mol) underargon, then TDAP (4.4 mL, 24 mmol, 20 mol %) was slowly added and thereactin mixture was stirred at 60° C. for 2 h. Product was destillatedof on Kugelrohr apparatus (125° C. at 0.1 mbar).

JAM0116

The same method of preparation as for previous compound JAM0149. Diethylphosphite (4.5 mL, 35 mmol), A solution of trimethylaluminium (2 M inhexanes, 17.5 mL, 35 mmol, 1.0 equiv.) JAM0112 (7.0 g, 35 mmol, 1.0equiv.) dichloromethane (120 mL). Filtration through pad of silica gel(hexane-ethyl acetate 1:1 to 3:1) Product JAM0116 (11 g, 94%) as an oil.1H NMR and 13C NMR spectra were in agreement with the published data.

JAM0117

The same method of preparation as for previous compound JAM0149. Diethylphosphite (2.7 mL, 21 mmol), A solution of trimethylaluminium (2 M inhexanes, 10.5 mL, 21 mmol, 1.0 equiv.) JAM0115 (4.8 g, 21 mmol, 1.0equiv.) dichloromethane (70 mL). Filtration through pad of silica gel(hexane-ethyl acetate 1:1 to 3:1) Product JAM0117 (7.2 g, 94%) as anoil. ¹H NMR (400 MHz, CDCl₃): 0.92 (3H, t, J=7.4), 0.94 (3H, t, J=7.4),1.28-1.32 (6H, m), 1.59-1.70 (4H, m), 1.79-1.89 (1H, m), 1.92-2.05 (2H,m), 2.19-2.40 (3H, m), 2.74-2.84 (1H, m), 4.00-4.12 (8H, m). ³¹P NMR(162 MHz, CDCl₃): 28.55

ESI MS: 389 (M+Na⁺).

HR ESI MS: calcd for C₁₆H₃₁O₂NaP 389.16996; found 389.16869.

JAM0151

Phosphonate JAM0149 (25.4 g, 60 mmol), was dissolved in dichloromethane(100 mL) and trifluoroacetic acid (100 mL) was slowly added. Thereaction mixture was stirred at room temperature overnight. Then thesolvents were removed in vacuo. The residue was filtered through a shortpad of silica gel (chloroform-methanol 10:1) to furnish the desiredproduct (19.7 g, 88%) as an oil.

ESI MS: 395 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅O₇NaP 395.12301; found 395.12337.

JAM0153

Dry flask was charged with phosphonate JAM0151 (1.95 g, 5.24 mmol), NaI(1.57 g, 7.86 mmol, 2 equiv.), triethylamine (1.1 mL, 7.86 mmol, 1.5equiv.). Dry DMF was added and reaction mixture was stirred at roomtemperature for 15 min. and then Chloromethyl pivalate (1.5 mL, 10.47mmol, 2 equiv.) was slowly added. Reaction mixture was stirred at roomtemperature overnight. The solvent was removed under reduced pressureand the residue was chromatographed on silica gel (ethyl acetate-hexane40:1) to afford the desired product (1.38 g, 54%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0162

The same method of preparation as for previous compound JAM0153.Phosphonate JAM0151 (2.15 g, 5.77 mmol), NaI (1.73 g, 11.55 mmol, 2equiv.), triethylamine (1.21 mL, 8.66 mmol, 1.5 equiv.), Chloromethylisopropyl carbonate (1.55 mL, 11.55 mmol, 2 equiv.), DMF (30 mL).Chromatography on silica gel (hexane-ethyl acetate 2:1). Product (1.97g, 70%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0182

The same method of preparation as for previous compound JAM0153.Phosphonate JAM0151 (2 g, 5.37 mmol), NaI (1.61 g, 10.74 mmol, 2equiv.), triethylamine (1.5 mL, 10.74 mmol, 2 equiv.), 1-Chloroethylisopropyl carbonate (1.64 mL, 10.74 mmol, 2 equiv.), DMF (30 mL).Chromatography on silica gel (hexane-ethyl acetate 1:1). Product (1.1 g,21%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0179

The same method of preparation as for previous compound JAM0153.Phosphonate JAM0151 (1.0 g, 2.68 mmol), NaI (805 mg, 5.37 mmol, 2equiv.), triethylamine (750 μL, 5.37 mmol, 2 equiv.),2-Chloro-N,N-dimethylacetamide (552 μL, 5.37 mmol, 2 equiv.), DMF (14mL). Chromatography on silica gel (chloroform-methanol 20:1). Product (1g, 85%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0154

The compound JAM0153 (3.3 g, 6.78 mmol) was dissolved in absolutedichloromethane (40 mL) under argon and cooled to 0° C. Abromotrimethylsilane (3.6 mL, 27.13 mmol, 4 equiv.) was added dropwiseand the solution was stirred at 0° C. overnight. The volatiles wereremoved in vacuo and the residue was diluted with mixture of methanoland toluene (3×30 mL, 1:1) and evaporated to obtain desired product(2.77 mg, 95%) as an oil and directly used in next reaction withoutcharacterization.

JAM0164

The same method of preparation as for previous compoundJAM0154.Phosphonate JAM0162 (1.6 g, 3.28 mmol), TMSBr (1.54 mL, 11.68mmol, 4 equiv.), DCM (20 mL). Product (1.39 g, 98%) obtained as an oil.

JAM0185

The same method of preparation as for previous compound JAM0154.Phosphonate JAM0182 (770 mg, 1.53 mmol), TMSBr (809 μL, 6.13 mmol, 4equiv.), DCM (10 mL). Product (669 mg, 98%) obtained as an oil.

JAM0184

The same method of preparation as for previous compound JAM0154.Phosphonate JAM0179 (1.05 g, 2.30 mmol), TMSBr (1.21 mL, 9.18 mmol, 4equiv.), DCM (13 mL). Product (904 mg, 98%) obtained as an oil.

JAM0167

Dry Schlank flask was charged with previous compound JAM0154 (600 mg,1.39 mmol) and dissolved in dry dioxane (7 mL). DBU (0.42 mL, 2.80 mmol,2 equiv), Chloromethyl pivalate (0.8 mL, 5.58 mmol, 4 equiv.) was addedand the reaction mixture was stirred at 100° C. for 6 h. The volatileswere removed in vacuo and the residue was chromatographed on silica gel(toluene-aceton 10:1 to afford impure desired product (48 mg, 5.2%) asan oil. The product was further purified using preparative scale HPLC(gradient 10:50, Rt=12.5 min.).

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0166

The same method of preparation as for previous compound JAM0167.Phosphonate JAM0164 (1.14 g, 2.64 mmol), DBU (0.79 mL, 5.28 mmol, 2equiv), Chloromethyl isopropyl carbonate (3.5 mL, 26.40 mmol, 10equiv.), dioxane (14 mL). Chromatography on silica gel (toluene-aceton5:1). Product (mg, <10%) as an oil. The product was further purifiedusing preparative scale HPLC (gradient 10:50, Rt=12.5 min.).

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0189

Dry Schlank flask was charged with previous compound JAM0185 (574 mg,1.29 mmol), K₂CO₃ (550 mg, 3.98 mmol, 3.1 equiv.) and dissolved in dryDMF (12 mL). 1-Chloroethyl isopropyl carbonate (2 mL, 12.86 mmol, 10equiv.) was added and the reaction mixture was stirred at 60° C. for 6h. The volatiles were removed in vacuo and the residue waschromatographed on silica gel (toluene-aceton 7:1) to afford impuredesired product (88 mg, 10%) as an oil. The product was further purifiedusing preparative scale HPLC (gradient 10:50, R_(t)=12.5 min.).

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0188

The same method of preparation as for previous compound JAM0167.Phosphonate JAM0184 (555 mg, 1.38 mmol), DBU (433 μL, 2.9 mmol, 2.1equiv), Chloromethyl isopropyl carbonate (1.85 mL, 13.82 mmol, 10equiv.), dioxane (7 mL). Chromatography on silica gel(chloroform-methanol 40:1). Product (96 mg, 11%) as an oil. The productwas further purified using preparative scale HPLC (gradient 10:50,Rt=12.5 min.).

ESI MS: 656 (M+Na⁺).

HR ESI MS: calcd for C₂₇H₄₀O₁₄NNaP 656.20786; found 656.20772

JAM0187

The same method of preparation as for previous compound JAM0189.Phosphonate JAM0184 (200 mg, 0.498 mmol), K₂CO₃ (344 mg, 2.49 mmol, 5equiv.), 1-Chloroethyl isopropyl carbonate (304 μL, 1.99 mmol, 2equiv.), DMF (6 mL). Chromatography on silica gel (chloroform-methanol10:1). Product (92 mg, 28%) as an oil. The product was further purifiedusing preparative scale HPLC (gradient 10:50, Rt=12.5 min.).

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805

JAM0168

The previous compound JAM0167 (48 mg, 72.9 mmol) was dissolved in dryTHF (3 mL). 10% Palladium on carbon (5 mg) was added and reactionmixture was bubbled with hydrogen for 10 min. Reaxtion misture wasstirred at room temperature overnight under hydrogen atmosphere.Palladium was filtered through cotton and the volatiles were removed invacuo to afford desired product (40 mg, 98%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0186

The same method of preparation as for previous compound JAM0168 (FIG.1). Phosphonate (1.18 g, 1.77 mmol), 10% palladium on carbon (100 mg),THF (70 mL). Product (996 mg, 98%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0195

The same method of preparation as for previous compound JAM0168.Phosphonate JAM0189 (1.0 g, 1.42 mmol), 10% palladium on carbon (100mg), THF (45 mL). Product (587 mg, 98%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805.

JAM0191

The same method of preparation as for previous compound JAM0168.Phosphonate JAM0188 (100 mg, 0.158 mmol), 10% palladium on carbon (10mg), THF (5 mL). Product (84 mg, 98%) as an oil.

ESI MS: 566 (M+Na⁺).

HR ESI MS: calcd for C₂₀H₃₄O₁₄NNaP 566.16091; found 566.16087

JAM0196

The same method of preparation as for previous compound JAM0168.Phosphonate JAM0187 (100 mg, 0.151 mmol), 10% palladium on carbon (10mg), THF (5 mL). Product (84 mg, 98%) as an oil.

ESI MS: 594 (M+Na⁺).

HR ESI MS: calcd for C₂₂H₃₈O₁₄NaP 594.19221; found 594.19215

JAM0214

A flask was charged with phosphonate JAM0151 (2.0 g, 5.37 mmol), DCC(1.22 g, 5.91 mmol, 1.1 equiv.), DMAP (65.6 mg, 0.54 mmol, 10 mol %).Dry dichloromethane was added and the reaction mixture was stirred atroom temperature for 15 min. Then 4-Acetamidophenol (974 mg, 6.44 mmol,1.2 equiv.) was added in one portion. Reaction mixture was stirred atroom temperature overnight. N,N-Dicyclohexylurea was filtered off andthe organic solvent was evaporated in vacuo. The residue waschromatographed on silica gel (chloroform-methanol 20:1) to afford thedesired product (1.55 g, 57%) as an oil.

ESI MS: 528 (M+Na⁺).

HR ESI MS: calcd for C₂₅H₃₂O₈NNaP 528.17577; found 528.17598

JAM0216

The same method of preparation as for previous compound JAM0154.Phosphonate JAM0214 (1.24 g, 2.48 mmol), TMSBr (1.31 mL, 9.92 mmol, 4equiv.), DCM (16 mL). Product (1.1 g, 98%) obtained as an oil. JAM0218

The same method of preparation as for previous compound JAM0189.Phosphonate JAM0216 (200 mg, 0.498 mmol), K₂CO₃ (344 mg, 2.49 mmol, 5equiv.), 1-Chloroethyl isopropyl carbonate (304 μL, 1.99 mmol, 2equiv.), DMF (6 mL). Chromatography on silica gel (chloroform-methanol10:1). Product (90 mg, 25%) as an oil. The product was further purifiedusing preparative scale HPLC (gradient 10:50, Rt=12.5 min.).

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805

JAM0219

The same method of preparation as for previous compound JAM0168.Phosphonate JAM0218 (100 mg, 0.158 mmol), 10% palladium on carbon (10mg), THF (5 mL). Product JAM0218 (92 mg, 98%) as an oil.

ESI MS: 323 (M+Na⁺).

HR ESI MS: calcd for C₁₇H₃₅NO₂SNa 340.22807; found 340.22805 NMR (400MHz, CDCl₃): 0.93 (3H, t, J=7.2), 0.96 (3H, t, J=7.2), 1.63 (2H, q,J=7.2), 1.7 (2H, q, J=7.2), 2.50-2.54 (2H, m), 2.62-2.66 (2H, m), 4.02(2H, t, J=6.7), 4.11 (2H, t, J=6.6), 5.58 (1H, d, J=1.2), 6.19 (1H, s).

¹³C NMR (126 MHz, CDCl₃): 10.35, 10.43, 21.99, 22.00, 27.41, 33.17,66.01, 66.30, 125.43, 139.25, 166.66, 172.71.

CI MS: 229 (M+W).

HR CI MS: calcd for C₁₂H₂₁O₄ 229.1440; found 229.1445.

5-benzyl 1-(((isopropoxycarbonyl)oxy)methyl) 2-((diphenoxyphosphoryl)methyl)pentanedioate JAM0338

The compound JAM0162 (400 mg, 0.819 mmol) was dissolved in absolutecooled to 0° C. A bromotrimethylsilane (0.30 mL, 3.28 mmol, 4 equiv.)was added dropwise and the solution was stirred at 0° C. overnight. Thevolatiles were removed in vacuo and the residue was diluted with mixtureof acetonitrile and water (5 mL, 4:1) and evaporated. The residue wasdissolved in absolute dichloromethane and catalytic amount of DMF (8 μL)was added. To the reaction mixture was added oxalyl chloride (0.480 mL,5.73 mmol, 7 equiv.) and the reaction mixture was stirred at roomtemperature for 2 h. The volatiles were removed in vacuo and the residuewas dissolved under argon in absolute dichloromethane (5 mL) and cooledto −20° C. To this mixture was added mixture of phenol (162 mg, 1.72mmol, 2.1 equiv.), diisopropylethylamine (0.5 mL) and pyridine (0.1 mL)in dichloromethane (3 mL). The reaction mixture was warmed slowly toroom temperature and then stirred for 12 h. The volatiles were removedin vacuo and the residue was chromatographed on silica gel (hexane-ethylacetate 2:1 to afford desired product (308 mg, 64%) as an oil. ¹H NMR(400 MHz, CDCl₃): 1.23 (3H, d, J=2.1), 1.25 (3H, d, J=2.1), 2.09-2.18(2H, m), 2.18-2.28 (1H, m), 2.39-2.52 (2H, m), 2.56-2.67 (1H, m),3.07-3.18 (1H, m), 4.85 (1H, hept, J=6.3), 5.11 (2H, s), 5.70 (1H, d,J=5.7), 5.77 (1H, d, J=5.7), 7.11-7.19 (6H, m), 7.27-7.38 (9H, m).

¹³C NMR (101 MHz, CDCl₃): 21.67 (2C), 27.94 (d, J_(C,P)=143.9), 28.19(d, J_(C,P)=12.4), 31.24, 39.18 (d, J_(C,P)=3.9), 66.63, 73.29, 82.28,120.58 (2C, d, J_(C,P)=2.3), 120.62 (2C, d, J_(C,P)=2.3), 125.42 (d,J_(C,P)=1.1), 125.44 (d, J_(C,P)=1.1), 128.39, 128.41, 128.68, 129.33(2C), 129.94 (2C), 135.82, 150.11 (d, J_(C,P)=3.4), 150.20 (d,J_(C,P)=3.6), 153.34, 172.13, 172.29 (d, J_(C,P)=9.3).

³¹P NMR (101 MHz, CDCl₃): 23.82

ESI MS: 607 ([M+Na]⁺).

HR ESI MS: calcd for C₃₀H₃₃O₁₀NaP 607.17035; found 607.17038.

4-((diphenoxyphosphoryl)methyl)-5-(((isopropoxycarbonyl)oxy)methoxy)-5-oxopentanoicAcid JAM0338H

The same method of preparation as for previous compound JAM0278R.Phosphonate (300 mg, 0.51 mmol), 10% palladium on carbon (10 mg), THF (5mL). Product (247 mg, 98%) as an oil. The product was further purifiedusing preparative scale HPLC.

¹H NMR (400 MHz, CDCl₃): 1.26 (3H, d, J=3.9), 1.28 (3H, d, J=3.8),2.04-2.20 (2H, m), 2.21-2.31 (1H, m), 2.37-2.51 (2H, m), 2.58-2.68 (1H,m), 3.09-3.19 (1H, m), 4.88 (1H, hept, J=6.3), 5.71 (1H, d, J=5.7), 5.80(1H, d, J=5.7), 7.12-7.19 (6H, m), 7.28-7.33 (4H, m).

¹³C NMR (101 MHz, CDCl₃): 21.68 (2C), 27.86 (d, J_(C,P)=144.1), 27.95(d, J_(C,P)=12.3), 30.86, 39.02 (d, J_(C,P)=3.8), 73.38, 82.31, 120.58(2C, d, J_(C,P)=1.9), 120.62 (2C, d, J_(C,P)=1.9), 125.54 (2C), 129.97(4C), 150.03 (d, J_(C,P)=3.4), 150.12 (d, J_(C,P)=3.6), 153.38, 172.25(d, J_(C,P)=9.5), 176.87.

³¹P NMR (101 MHz, CDCl₃): 24.00

ESI MS: 517 ([M+Na]⁺).

HR ESI MS: calcd for C₂₃H₂₈O₁₀P 495.14146; found 495.14111.

5-benzyl 1-(((isopropoxycarbonyl)oxy)methyl)2-((bis(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)methyl)pentanedioateJAM0341

The compound JAM0162 (400 mg, 0.819 mmol) was dissolved in absolutedichloromethane (5 mL) under argon and cooled to 0° C. Abromotrimethylsilane (0.30 mL, 3.28 mmol, 4 equiv.) was added dropwiseand the solution was stirred at 0° C. overnight. The volatiles wereremoved in vacuo and the residue was diluted with mixture ofacetonitrile and water (5 mL, 4:1) and evaporated. The residue wasdissolved in absolute dichloromethane and catalytic amount of DMF (8 μL)was added. To the reaction mixture was added oxalyl chloride (0.480 mL,5.73 mmol, 7 equiv.) and the reaction mixture was stirred at roomtemperature for 2 h. The volatiles were removed in vacuo and the residuewas dissolved under argon in absolute dichloromethane (5 mL) and cooledto −20° C. To this mixture was added mixture of L-alanine ethyl esterhydrochloride (264 mg, 1.72 mmol, 2.1 equiv.), diisopropylethylamine(1.0 mL) and pyridine (0.1 mL) in dichloromethane (3 mL). The reactionmixture was warmed slowly to room temperature and then stirred for 12 h.The volatiles were removed in vacuo and the residue was chromatographedon silica gel (chloroform-acetone 5:1 to afford desired product (238 mg,46%) as an oil.

¹H NMR (400 MHz, CDCl₃): 1.15-1.20 (12H, m), 1.27-1.32 (6H, m),1.67-1.80 (1H, m), 1.88-2.02 (1H, m), 2.08-2.21 (1H, m), 2.31-2.36 (2H,m), 2.83-2.98 (1H, m), 3.07-3.24 (2H, m), 3.85-3.98 (2H, m), 4.00-4.12(4H, m), 4.80 (1H, hept, J=6.3), 5.02 (2H, s), 5.66 (1H, dd, J=43.8,5.6), 5.70 (1H, dd, J=67.6, 5.7), 7.21-7.29 (5H, m).

³¹P NMR (101 MHz, CDCl₃): 28.32 and 28.38 (mixture of diastereoizomers)

4-((bis(((S)-1-ethoxy-1-oxopropan-2-yl)amino)phosphoryl)methyl)-5-(((isopropoxycarbonyl)oxy)methoxy)-5-oxopentanoicAcid JAM0341H

The same method of preparation as for previous compound JAM0278R.Phosphonate (100 mg, 0.16 mmol), 10% palladium on carbon (5 mg), THF (4mL). Product (84 mg, 98%) as an oil. The product was further purifiedusing preparative scale HPLC.

¹H NMR (400 MHz, CDCl₃): 1.23-1.30 (12H, m), 1.34-1.38 (6H, m),1.86-2.02 (3H, m), 2.16-2.27 (1H, m), 2.28-2.41 (2H, m), 2.93-3.04 (1H,m), 3.41-3.60 (2H, m), 3.89-4.02 (2H, m), 4.09-4.21 (4H, m), 4.89 (1H,hept, J=6.2), 5.74 (1H, dd, J=41.2, 5.7), 5.76 (1H, dd, J=53.7, 5.7).

³¹P NMR (101 MHz, CDCl₃): 31.00 (both diastereoizomers)

ESI MS: 563 ([M+Na]⁺).

HR ESI MS: calcd for C₂₁H₃₇O₁₂N₂NaP 563.19763; found 563.19765.

Allyl-3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propanoate LTP089

Meldrum's acid (5.0 g, 34.69 mmol, 1 eq.), a freshly grinded K₂CO₃ (4.8g, 34.69 mmol, 1 eq.) and BnEt₃NCl (7.9 g, 34.69 mmol, 1 eq.) weresuspended in dry AcN (50 mL). The reaction mixture was stirred for 1hour at room temperature under inert atmosphere. Allyl acrylate (5.8 g,6.2 mL, 52.04 mmol, 1.5 eq.) was added and the mixture was heated to 65°C. for 22 hours. AcN was evaporated. The residue was dissolved in EtOAc(200 mL), extracted with 10% KHSO₄ (3×150 mL), dried over MgSO₄ and thesolvent was removed by vacuo. The crude product was suspended in hexane(50 mL) and sonicated. Desired product (7.3 g, 82%) was obtained afterfiltration as a colorless solid compound.

¹H NMR (400 MHz, CDCl₃): 1.76 (3H, s), 1.81 (3H, s), 2.39 (2H, dt,J=7.2, 5.6), 2.67 (2H, t, J=7.2), 3.91 (1H, t, J=5.6), 4.57 (2H, dt,J=5.8, 1.4), 5.23 (1H, dq, J=10.4, 1.3), 5.30 (1H, dq, J=17.2, 1.5),5.90 (1H, ddt, J=17.2, 10.4, 5.8).

¹³C NMR (101 MHz, CDCl₃): 21.30, 26.54, 28.66, 30.33, 44.84, 65.43,105.23, 118.60, 132.05, 165.23 (2C), 172.61.

T-Butyl-3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propanoate LTP096

Meldrum's acid (10.0 g, 69.38 mmol, 1 eq.), a freshly grinded K₂CO₃ (9.6g, 69.38 mmol, 1 eq.) and BnEt₃NCl (15.8 g, 69.38 mmol, 1 eq.) weresuspended in dry AcN (100 mL). The reaction mixture was stirred for 1hour at room temperature under inert atmosphere. t-Butyl acrylate (13.3g, 15.1 mL, 104.07 mmol, 1.5 eq.) was added and the mixture was heatedto 65° C. for 22 hours. AcN was evaporated. The residue was dissolved inEtOAc (300 mL), extracted with 10% KHSO₄ (3×200 mL), dried over MgSO₄and the solvent was removed by vacuo. The crude product was suspended inhexane (100 mL) and sonicated. Desired product (11.4 g, 60%) wasobtained after filtration as a colorless solid compound.

¹H NMR (400 MHz, CDCl₃): 1.42 (9H, s), 1.75 (3H, s), 1.80 (3H, s),2.28-2.38 (2H, m), 2.52 (2H, td, J=7.3, 0.6), 3.91 (1H, t, J=5.6).

¹³C NMR (101 MHz, CDCl₃): 21.47, 26.57, 28.20 (3C), 28.67, 31.52, 44.93,80.98, 105.12, 165.35 (2C), 172.25.

Diallyl-2-methylenepentanedioate LTP013

A dry Schlenk flask was charged with allyl acrylate (1.00 g, 8.92 mmol,2 eq.). Tributylphosphine (217 mg, 267 μL, 0.24 eq.) was added by dropwise. The reaction mixture was stirred for 2 h at rt under inert(exothermic at the beginning of reaction).

The crude product was purified by column chromatography (hexane-ethylacetate 15:1) and the desired product was obtained as a colourless oil(785 mg) in 79% yield.

¹H NMR (400 MHz, CDCl₃): 2.54-2.59 (2H, m), 2.67 (2H, t, J=7.4), 4.57(2H, dt, J=5.8, 1.4), 4.66 (2H, dt, J=5.8, 1.4), 5.15-5.38 (4H, m), 5.62(1H, d, J=1.2), 5.84-6.01 (2H, m), 6.23 (1H, d, J=1.2).

¹³C NMR (101 MHz, CDCl₃): 27.45, 33.19, 65.31, 65.54, 118.31, 118.43,126.30, 132.21, 132.27, 138.93, 166.39, 172.45.

5-Allyl-1-t-butyl-2-methylenepentanedioate LTP091

A dry Schlenk flask was charged with the compound LTP089 (4.00 g, 15.61mmol), N,N-Dimethylmethyleneiminium iodide (7.22 g, 39.02 mmol, 2.5 eq.)and then it was flushed with argon. Absolute t-BuOH (100 mL) was addedto the flask and the mixture was stirred at 70-75° C. for 20 h. Theorganic solvent was evaporated in vacuo. The residue was dissolved inEt₂O (200 mL) and extracted with sat. NaHCO₃ (150 mL), 10% KHSO₄ (150mL), 10% Na₂S₂O₅ (150 mL), sat. NaCl (150 mL), dried over MgSO₄. Solventwas evaporated. The crude product was purified by column chromatography(hexane-ethyl acetate 8:1) to afford the desired product (3.04 g, 81%)as a colourless oil.

¹H NMR (400 MHz, CDCl₃): 1.48 (9H, s), 2.49-2.55 (2H, m), 2.56-2.64 (2H,m), 4.56 (2H, dt, J=5.7, 1.4), 5.21 (1H, dt, J=10.4, 1.2), 5.29 (1H, dt,J=10.4, 1.2), 5.49 (1H, d, J=1.2), 5.83-5.95 (1H, m). 6.08 (1H, d,J=1.2).

¹³C NMR (101 MHz, CDCl₃): 27.53, 28.17, 33.29, 65.22, 80.88, 118.30,124.87, 132.30, 140.57, 165.99, 172.56.

1-Allyl-5-t-butyl-2-methylenepentanedioate LTP097

A dry Schlenk flask was charged with the compound LTP096 (5.00 g, 18.36mmol), N,N-Dimethylmethyleneiminium iodide (8.49 g, 45.91 mmol, 2.5 eq.)and then it was flushed with argon. Absolute allyl alcohol (50 mL) wasadded to the flask and the mixture was stirred at 70° C. for 20 h. Theorganic solvent was evaporated in vacuo. The residue was dissolved inEt₂O (200 mL) and extracted with sat. NaHCO₃ (150 mL), 10% KHSO₄ (150mL), 10% Na₂S₂O₅ (150 mL), sat. NaCl (150 mL), dried over MgSO₄. Solventwas evaporated. The crude product was purified by column chromatography(hexane-ethyl acetate 10:1) to afford the desired product (2.37 g, 54%)as a colourless oil.

¹H NMR (400 MHz, CDCl₃): 1.43 (9H, s), 2.38-2.48 (2H, m), 2.56-2.66 (2H,m), 4.56 (2H, dt, J=5.6, 1.5), 5.24 (1H, dt, J=10.4, 1.3), 5.33 (1H, dt,J=17.2, 1.6), 5.60 (1H, d, J=1.3), 5.95 (1H, ddt, J=17.2, 10.4, 5.6).6.21 (1H, d, J=1.3).

¹³C NMR (101 MHz, CDCl₃): 27.57, 28.23, 34.35, 65.47, 80.57, 118.20,125.87, 132.28, 139.23, 166.51, 172.13.

Diallyl-2-((diethoxyphosphoryl)methyl)pentanedioate LTP016

Diethyl phosphite (439 mg, 410 μL, 3.18 mmol, 1 eq.) was dissolved inabsolute dichloromethane (8 mL) under argon and cooled to 0° C. Asolution of trimethyl aluminium (2 M in hexanes, 1.59 mL, 3.18 mmol, 1equiv.) was added dropwise and the solution was stirred at 0° C. for 30min. The solution of the compound LTP013 (712 mg, 3.18 mmol, 1 eq.) indichloromethane (3 mL) was added during 10 min at 0° C., the mixture wasstirred next 30 min at the same temperature and then the cooling bathwas removed. The reaction mixture was stirred at room temperatureovernight. After 16 h the reaction was quenched with 2 N hydrochloricacid (10 mL). The organic layer was separated and a water phase wasextracted with DCM (2×20 mL). The combined organic layers were washedwith water (20 mL), brine (20 mL) and dried over anhydrous MgSO₄. Thecrude product was an oil, which was filtered through pad of silica gel(EtOAc) to afford the desired product (1.04 g, 90%) as a colourless oil.

¹H NMR (400 MHz, CDCl₃): 1.24-1.28 (6H, m), 1.81 (1H, ddd, J=18.5, 15.4,5.3), 1.90-2.02 (2H, m), 2.21 (1H, ddd, J=18.1, 15.5, 8.5), 2.27-2.39(2H, m), 2.79 (1H, tdd, J=13.6, 8.3, 5.5), 4.03 (4H, tt, J=8.7, 5.2),4.53 (4H, dd, J=12.1, 5.7), 5.18 (2H, ddd, J=10.4, 6.8, 1.4), 5.27 (2H,ddd, J=17.2, 12.6, 1.3), 5.79-5.93 (2H, m).

5-Allyl-1-t-butyl-2-((diethoxyphosphoryl)methyl)pentanedioate LTP093

Diethyl phosphite (1.72 g, 1.60 mL, 12.44 mmol, 1 eq.) was dissolved inabsolute dichloromethane (30 mL) under argon and cooled to 0° C. Asolution of trimethyl aluminium (2 M in hexanes, 6.22 mL, 12.44 mmol, 1equiv.) was added dropwise and the solution was stirred at 0° C. for 30min. The solution of the compound LTP091 (2.99 g, 12.44 mmol, 1 eq.) indichloromethane (10 mL) was added during 15 min at 0° C., the mixturewas stirred next 30 min at the same temperature and then the coolingbath was removed. The reaction mixture was stirred at room temperatureovernight. After 20 h the reaction was quenched with 2 N hydrochloricacid (40 mL). The organic layer was separated and a water phase wasextracted with DCM (2×50 mL). The combined organic layers were washedwith water (20 mL), brine (20 mL) and dried over anhydrous MgSO₄. Thecrude product was an oil, which was filtered through pad of silica gel(EtOAc-hexane 2:1) to afford the desired product (4.13 g, 88%) as acolourless oil. ¹H NMR (400 MHz, CDCl₃): 1.28 (6H, td, J=7.1, 1.7), 1.42(9H, s), 1.69-1.80 (1H, m), 1.84-2.00 (2H, m), 2.12-2.24 (1H, m),2.26-2.45 (2H, m), 2.59-2.73 (1H, m), 4.06 (4H, dq, J=8.2, 7.1), 4.54(2H, dt, J=5.7, 1.4), 5.19 (1H, dq, J=10.4, 1.3), 5.27 (1H, dq, J=17.2,1.5), 5.82-5.93 (1H, m).

1-Allyl-5-t-butyl-2-((diethoxyphosphoryl)methyl)pentanedioate LTP100

Diethyl phosphite (1.36 g, 1.27 mL, 9.86 mmol, 1 eq.) was dissolved inabsolute dichloromethane (26 mL) under argon and cooled to 0° C. Asolution of trimethyl aluminium (2 M in hexanes, 4.93 mL, 9.86 mmol, 1equiv.) was added dropwise and the solution was stirred at 0° C. for 30min. The solution of the compound LTP097 (2.37 g, 9.86 mmol, 1 eq.) indichloromethane (9 mL) was added during 15 min at 0° C., the mixture wasstirred next 30 min at the same temperature and then the cooling bathwas removed. The reaction mixture was stirred at room temperatureovernight. After 16 h the reaction was quenched with 2 N hydrochloricacid (35 mL). The organic layer was separated and a water phase wasextracted with DCM (2×45 mL). The combined organic layers were washedwith water (20 mL), brine (20 mL) and dried over anhydrous MgSO₄. Thecrude product was an oil, which was filtered through pad of silica gel(EtOAc-hexane 2:1) to afford the desired product (3.20 g, 86%) as acolourless oil.

¹H NMR (400 MHz, CDCl₃): 1.29 (6H, tdd, J=7.1, 3.3, 0.4), 1.42 (9H, s),1.78-1.88 (1H, m), 1.89-1.99 (2H, m), 2.16-2.33 (3H, m), 2.75-2.86 (1H,m), 4.01-4.12 (4H, m), 4.59 (2H, dq, J=5.8, 1.2), 5.23 (1H, dq, J=10.4,1.3), 5.32 (1H, dq, J=17.2, 1.5), 5.91 (1H, ddt, J=17.2, 10.4, 5.8).

¹³C NMR (101 MHz, CDCl₃): 16.47 (d, J_(C,P)=2.0), 16.53 (d,J_(C,P)=1.8), 37.91 (d, J_(C,P)=142.6), 28.18, 28.82 (d, J_(C,P)=13.0),32.78, 39.37, 61.83 (d, J_(C,P)=6.5), 61.92 (d, J_(C,P)=6.3), 65.66,80.66, 118.63, 171.87, 173.92 (d, J_(C,P)=7.9).

5-(Allyloxy)-2-((diethoxyphosphoryl)methyl)-5-oxopentanoic acid LTP111

Phosphonate LTP093 (1.50 g, 3.96 mmol), was dissolved in dichloromethane(15 mL) and the mixture was cooled to 0° C. Trifluoroacetic acid (15 mL)was added slowly during 15 minutes. The reaction mixture was stirred atroom temperature overnight (18 h). Then the solvents were removed invacuo. The residue was dissolved in PhCH₃ (2×15 mL) and evaporated. Thecrude product was purified on silica gel (chloroform-methanol 12:1) tofurnish the desired product (1.22 g, 95%) as an oil.

¹H NMR (400 MHz, CDCl₃): 1.31 (6H, td, J=7.1, 2.1), 1.80-2.05 (3H, m),2.25-2.49 (3H, m), 2.78-2.92 (1H, m), 4.05-4.17 (4H, m), 4.59 (2H, dq,J=5.9, 1.3), 5.25 (1H, dq, J=10.4, 1.2), 5.33 (1H, dq, J=17.2, 1.5),5.90 (1H, ddt, J=17.2, 10.4, 5.9), 11.40 (1H, bs).

¹³C NMR (101 MHz, CDCl₃): 16.31 (2C, d, J_(C,P)=6.2), 27.43 (d,J_(C,P)=131.9), 28.21, 31.19, 39.02 (d, J_(C,P)=3.7), 62.97 (d,J_(C,P)=5.4), 63.03 (d, J_(C,P)=5.1), 65.98, 119.07, 131.76, 173.48 (d,J_(C,P)=8.5), 177.84.

5-(Allyloxy)-4-((diethoxyphosphoryl)methyl)-5-oxopentanoic acid LTP104

Phosphonate LTP100 (3.196 g, 8.43 mmol), was dissolved indichloromethane (30 mL) and the mixture was cooled to 0° C.Trifluoroacetic acid (30 mL) was added slowly during 20 minutes. Thereaction mixture was stirred at room temperature overnight (16 h). Thenthe solvents were removed in vacuo. The residue was dissolved in PhCH₃(2×15 mL) and evaporated. The crude product was purified on silica gel(chloroform-methanol 12:1) to furnish the desired product (2.60 g, 96%)as an oil.

¹H NMR (400 MHz, CDCl₃): 1.30 (6H, td, J=7.0, 2.1), 1.83-2.07 (3H, m),2.23-2.36 (1H, m), 2.37-2.49 (2H, m), 2.75-2.87 (1H, m), 4.04-4.19 (4H,m), 4.56 (2H, dt, J=5.8, 1.4), 5.21 (1H, dq, J=10.4, 1.2), 5.29 (1H, dq,J=17.2, 1.5), 5.88 (1H, ddt, J=17.2, 10.4, 5.8), 12.11 (1H, bs).

¹³C NMR (101 MHz, CDCl₃): 16.21 (d, J_(C,P)=2.6), 16.27 (d,J_(C,P)=2.6), 27.30 (d, J_(C,P)=144.2), 28.15 (d, J_(C,P)=12.6), 31.35,38.99 (d, J_(C,P)=3.7), 62.99 (d, J_(C,P)=2.5), 63.06 (d, J_(C,P)=2.4),65.48, 118.52, 132.03, 172.39, 178.38 (d, J_(C,P)=8.3).

(5-(Allyloxy)-2-((allyloxy)carbonyl)-5-oxopentyl)phosphonic acid LTP018

The compound LTP016 (4.02 g, 11.09 mmol) was dissolved in absolutedichloromethane (62 mL) under argon and cooled to 0° C.Bromotrimethylsilane (6.79 g, 5.86 mL, 44.38 mmol, 4 eq.) was addeddropwise during 20 min and the solution was stirred at 0° C. overnight.The volatiles were removed in vacuo and the residue was diluted withmixture of acetonitrile and water (30 mL, 4:1) and evaporated. The crudeproduct was obtained in quantitative yield (3.40 g) and it was used forthe next step without purification.

¹H NMR (400 MHz, CDCl₃): 1.84-2.02 (3H, m), 2.21-2.36 (1H, m), 2.37-2.43(2H, m), 2.79-2.92 (1H, m), 4.55-4.64 (414, m), 5.20-5.27 (2H, m),5.27-5.32 (1H, m), 5.32-5.37 (1H, m), 5.84-5.97 (2H, m), 9.89 (2H, bs).

5-(Allyloxy)-5-oxo-2-(phosphonomethyl)pentanoic acid LTP116

The compound LTP111 (500 mg, 1.55 mmol) was dissolved in absolutedichloromethane (10 mL) under argon and cooled to 0° C.Bromotrimethylsilane (1.43 g, 1.23 mL, 9.31 mmol, 6 eq.) was addeddropwise during 20 min and the solution was stirred at 0° C. overnight.The volatiles were removed in vacuo and the residue was diluted withmixture of acetonitrile and water (20 mL, 4:1) and evaporated. The crudeproduct was obtained in quantitative yield (413 mg) and it was used forthe next step without purification.

¹H NMR (400 MHz, d⁶-DMSO): 1.56-2.00 (3H, m), 2.24-2.42 (2H, m),2.52-2.61 (2H, m), 4.53 (2H, dt, J=5.4, 1.6), 5.20 (1H, dq, J=10.3,1.4), 5.29 (1H, dq, J=17.3, 1.7), 5.90 (1H, ddt, J=17.3, 10.3, 5.4),7.53 (3H, bs).

¹³C NMR (101 MHz, d⁶-DMSO): 27.62 (d, J_(C,P)=9.6), 29.48 (d,J_(C,P)=137.5), 31.09, 31.26, 64.43, 117.74, 132.78, 172.02, 175.55 (d,J_(C,P)=10.6).

5-(Allyloxy)-5-oxo-4-(phosphonomethyl)pentanoic acid LTP115

The compound LTP104 (500 mg, 1.55 mmol) was dissolved in absolutedichloromethane (10 mL) under argon and cooled to 0° C.Bromotrimethylsilane (1.43 g, 1.23 mL, 9.31 mmol, 6 eq.) was addeddropwise during 20 min and the solution was stirred at 0° C. overnight.The volatiles were removed in vacuo and the residue was diluted withmixture of acetonitrile and water (20 mL, 4:1) and evaporated. The crudeproduct was obtained in quantitative yield (413 mg) and it was used forthe next step without purification.

¹H NMR (400 MHz, P-DMSO): 1.59-1.81 (2H, m), 1.82-1.99 (2H, m),2.11-2.28 (2H, m), 2.61-2.72 (1H, m), 4.53 (2H, dt, J=5.6, 1.5), 5.20(1H, dq, J=10.5, 1.4), 5.31 (1H, dq, J=17.3, 1.7), 5.91 (1H, ddt,J=17.3, 10.5, 5.6), 7.09 (3H, bs).

¹³C NMR (101 MHz, d⁶-DMSO): 28.10 (d, J_(C,P)=11.2), 29.72 (d,J_(C,P)=137.4), 31.12 (2C), 64.67, 117.85, 132.76, 173.72, 173.80 (d,J_(C,P)=9.1).

Diallyl-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)pentanedioateLTP021

Compound LTP018 (104 mg, 0.340 mmol, 1 eq.) was dissolved in dry THF(1.5 mL). Triphenylphosphine (223 mg, 0.849 mmol, 2.5 eq.) and4-acetoxybenzyl alcohol (141 mg, 0.849 mmol, 2.5 eq.) was added in oneportion and finally DIAD (172 mg, 167 μL, 0.849 mmol, 2.5 eq.) was addedby dropwise during 5 min (exothermic reaction). The reaction mixture wasstirred for 1 h at rt. THF was evaporated by rotavap and the crudeproduct was purified by column chromatography (DCM/EtOAc 2:1). Thedesired product (150 mg, 73%) was obtained as a viscous colourless oil.

¹H NMR (400 MHz, CDCl₃): 1.81-2.01 (2H, m), 2.29 (6H, s), 2.30-2.40 (4H,m), 2.75-2.90 (1H, m), 4.37-4.52 (2H, m), 4.55 (2H, d, J=5.4), 4.85-5.06(4H, m), 5.16-5.42 (4H, m), 5.74-6.01 (2H, m), 7.07 (4H, dd, J=8.6,2.3), 7.35 (4H, dd, J=7.3, 2.5).

1-(4-Acetoxybenzyl)-5-allyl-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl) Pentanedioate LTP119

The compound LTP116 (300 mg, 1.13 mmol, 1 eq.) was dissolved in dry THF(9 mL). Triphenylphosphine (1.18 g, 4.51 mmol, 4 eq.) and4-acetoxybenzyl alcohol (749 mg, 4.51 mmol, 4 eq.) was added in oneportion and finally DIAD (912 mg, 888 μL, 4.51 mmol, 4 eq.) was added bydropwise during 5 min (exothermic reaction). The reaction mixture wasstirred for 1 h at rt. THF was evaporated by rotavap and the crudeproduct was purified by column chromatography (EtOAc/hexane 2:1 toEtOAc). The desired product (275 mg, 34%) was obtained as a viscouscolourless oil.

¹H NMR (400 MHz, CDCl₃): 1.82-2.02 (3H, m), 2.25-2.39 (3H, m), 2.30 (3H,s), 2.31 (6H, s), 2.79-2.91 (1H, m), 4.55 (2H, dt, J=5.7, 1.4),4.88-5.04 (6H, m), 5.22 (1H, dq, J=10.4, 1.3), 5.29 (1H, dq, J=17.2,1.5), 5.89 (1H, ddt, J=17.2, 10.4, 5.7), 7.03-7.11 (6H, m), 7.29-7.37(6H, m).

5-(4-Acetoxybenzyl)-1-allyl-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl) pentanedioate LTP122

Compound LTP122 (400 mg, 1.50 mmol, 1 eq.) was dissolved in dry THF (12mL). Triphenylphosphine (1.58 g, 6.01 mmol, 4 eq.) and 4-acetoxybenzylalcohol (999 mg, 6.01 mmol, 4 eq.) was added in one portion and finallyDIAD (1.22 g, 1.18 mL, 6.01 mmol, 4 eq.) was added by dropwise during 5min (exothermic reaction). The reaction mixture was stirred for 1 h atrt. THF was evaporated by rotavap and the crude product was purified bycolumn chromatography (EtOAc). The desired product (1.0 g, 94%) wasobtained as a viscous colourless oil.

¹H NMR (400 MHz, CDCl₃): 1.76-2.00 (3H, m), 2.22-2.38 (3H, m), 2.27 (3H,s), 2.28 (6H, s), 2.73-2.86 (1H, m), 4.43 (2H, dt, J=5.8, 1.6),4.85-5.02 (6H, m), 5.17 (1H, dq, J=10.3, 1.2), 5.24 (1H, dq, J=17.2,1.5), 5.89 (1H, ddt, J=17.2, 10.3, 5.8), 7.02-7.09 (6H, m), 7.28-7.38(6H, m).

¹³C NMR (101 MHz, CDCl₃): 21.05, 21.11 (2C), 21.68, 21.90, 21.96, 28.15(d, J_(C,P)=142.2), 28.39 (d, J_(C,P)=13.5), 31.32, 39.11 (d,J_(C,P)=3.7), 64.53, 65.64, 65.71, 66.74 (d, J_(C,P)=2.5), 66.80 (d,J_(C,P)=2.1), 69.99, 118.63, 121.59, 121.73, 121.81 (d, J_(C,P)=2.5),128.00, 129.24, 129.49, 131.79, 133.42, 133.63 (d, J_(C,P)=1.8), 133.69(d, J_(C,P)=1.4), 138.73, 149.95, 150.53, 150.71 (d, J_(C,P)=1.7),169.30, 169.40, 169.58, 172.14, 173.40 (d, J_(C,P)=7.8).

2-((Bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)pentanedioic acid LTP023

The starting material LTP021 (144 mg, 0.239 mmol, 1 eq.) was dissolvedin dry THF (2 mL). Pd(PPh₃)₄ (13.8 mg, 0.012 mmol, 5 mol %) was added inone portion and finally phenyl silane (103 mg, 117 μL, 0.956 mmol, 4eq.) was added by dropwise during 2 min. The reaction mixture wasstirred at rt under inert atmosphere for 1 h. THF was evaporated andcrude product was filtered through pad of silica gel (EtOAc/MeOH 2:1)and finally purified by preparative HPLC. The desired product (50 mg,40%) was obtained as an amorphous solid (after lyophilisation).

¹H NMR (400 MHz, CDCl₃): 1.70-2.04 (2H, m), 2.28 (6H, s), 2.31-2.49 (4H,m), 2.70-2.81 (1H, m), 4.85-5.04 (4H, m), 7.05 (4H, dd, J=8.4, 1.6),7.32 (4H, dd, J=8.5, 1.6), 7.81 (2H, s).

5-(4-Acetoxybenzyl)-4-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)-5-oxopentanoicAcid LTP120

The starting material LTP119 (139 mg, 0.196 mmol, 1 eq.) was dissolvedin dry THF (2 mL). Pd(PPh₃)₄ (11.3 mg, 0.010 mmol, 5 mol %) was added inone portion and finally phenyl silane (42 mg, 48 μL, 0.391 mmol, 2 eq.)was added by dropwise during 2 min. The reaction mixture was stirred atrt under inert atmosphere for 1 h. THF was evaporated and crude productwas filtered through pad of silica gel (CHCl₃/MeOH 20:1 to 10:1) andfinally purified by preparative HPLC. The desired product (114 mg, 87%)was obtained as an oil.

¹H NMR (400 MHz, CDCl₃): 1.82-1.97 (2H, m), 2.29 (3H, s), 2.30 (6H, s),2.16-2.37 (4H, m), 2.72-2.85 (1H, m), 4.86-5.03 (6H, m), 6.03 (1H, bs),7.00-7.08 (6H, m), 7.28-7.35 (6H, m).

¹³C NMR (101 MHz, CDCl₃): 21.25 (3C), 27.97 (d, J_(C,P)=142.8), 28.13(d, J_(C,P)=13.1), 30.80, 38.98 (d, J_(C,P)=3.6), 66.35, 67.32 (d,J_(C,P)=3.7), 67.38 (d, J_(C,P)=3.6), 121.88 (2C), 122.05 (2C), 122.06(2C), 129.55 (2C), 129.60 (2C), 129.78 (2C), 133.22, 133.51, 133.57,150.73, 150.91, 150.93, 169.59, 169.67, 169.74, 173.51 (d, J_(C,P)=8.5),175.36.

5-(4-Acetoxybenzyl)-2-((bis((4-acetoxybenzyl)oxy)phosphoryl)methyl)-5-oxopentanoicAcid LTP123

The starting material LTP122 (500 mg, 0.703 mmol, 1 eq.) was dissolvedin dry THF (4 mL). Pd(PPh₃)₄ (41 mg, 0.035 mmol, 5 mol %) was added inone portion and finally phenyl silane (152 mg, 173 μL, 1.41 mmol, 2 eq.)was added by dropwise during 2 min. The reaction mixture was stirred atrt under inert atmosphere for 1 h. THF was evaporated and crude productwas filtered through pad of silica gel (CHCl₃/MeOH 15:1) and finallypurified by preparative HPLC. The desired product (246 mg, 52%) wasobtained as an oil.

¹H NMR (400 MHz, CDCl₃): 1.83-2.04 (2H, m), 2.27 (6H, s), 2.28 (3H, s),2.30-2.47 (4H, m), 2.72-2.83 (1H, m), 4.88-5.03 (4H, m), 5.05 (2H, s),7.05 (6H, dd, J=8.5, 2.0), 7.32 (6H, dd, J=8.1, 1.4), 9.24 (1H, bs).

¹³C NMR (101 MHz, CDCl₃): 21.14 (2C), 21.15, 27.89 (d, J_(C,P)=143.0),28.22 (d, J_(C,P)=13.8), 31.35, 38.79 (d, J_(C,P)=3.5), 65.79, 67.47,67.54, 121.77 (2C), 121.94 (4C), 129.43 (2C), 129.44 (2C), 129.52 (2C),133.32, 133.38, 133.49, 150.56, 150.85 (2C), 169.52, 169.53, 169.57,172.40, 176.57 (d, J_(C,P)=7.5).

1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl) 5-(2-(trimethylsilyl)ethyl)2-((diethoxyphosphoryl)methyl)pentanedioate TT-160614

To a solution of2-((diethoxyphosphoryl)methyl)-5-oxo-5-(2-(trimethylsilyl)ethoxy)pentanoicacid (1.49 g, 3.90 mmol), 4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one(663 mg, 5.1 mmol) and HOBt (690 mg, 5.1 mmol) in anhydrous DMF (10 ml)a solution of EDC.HCl (980 mg, 5.1 mmol) and DMAP (623 mg, 5.1 mmol) inDMF (10 ml) was added. The mixture was stirred at RT overnight. Thesolvent was evaporated and the residue was extracted with EtOAc-H2Omixture (1:1, 200 ml). Aqueous portion was extracted with EtOAc (50 ml)again. Organic extracts were dried with MgSO4 and evaporated. Theresidue was chromatographed on a silica gel column in EtOAc→1%MeOH/EtOAc. Yield 1.75 g of oil (91%).

¹H NMR (400 MHz, CDCl₃): 0.04 (s, 9H, Si—CH₃); 0.98 (m, 2H, Si—CH₂);1.29-1.33 (m, 6H, CH₃(Et)), 1.88 (ddd, 1H, J_(1b-P)=18.7, J_(gem)=15.4,J_(1b-2)=5.0, H-1b), 1.95-2.03 (m, 2H, H-3), 2.19 (t, 3H,J_(CH3-CH2)=0.5, 4′—CH₃), 2.21 (ddd, J_(1a-P)=17.8, J_(gem)=15.4,J_(1a-2)=8.9, H-1a), 2.27-2.35 (m, 2H, H-4), 2.84 (m, 1H, H-2),4.05-4.12 (m, 4H, CH₂ (Et)), 4.16 (m, 2H, OCH₂(TMSE)), 4.84 (dq, 1H,J_(gem)=13.9, J_(CH2-CH3)=0.5, 3′—CH₂b), 4.89 (dq, 1H, J_(gem)=13.9,J_(CH2-CH3)=0.5, 3. —CH₂a).

¹³C NMR (101 MHz, CDCl₃): −1.55 (Si—CH₃), 9.37 (4′—CH₃), 16.37 (d,J_(CH3-P)=6.0, CH₃(Et)), 17.26 (Si—CH₂), 27.69 (d, J_(1-P)=128.7, C-1),28.31 (C-3), 31.41 (C-4), 39.15 (d, J_(2-P)=3.7, C-2), 54.17 (3′—CH₂),61.81-61.92 (m, CH₂ (Et)), 62.90 (OCH₂ (TMSE)), 133.24 (C-3′), 140.20(C-4′), 152.01 (C-1′), 171.40 (C-5), 173.48 (d, J_(C-2-1-P)=7.7, COO).

ESI MS: 517 ([M+Na]⁺).

HR ESI MS: calcd for C₂₀H₃₅O₁₀NaPSi 517.16293; found 517.16306.

1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl) 5-(2-(trimethylsilyl)ethyl)2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pentanedioateTT-230614

To a solution of 1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)5-(2-(trimethylsilyl)ethyl) 2-((diethoxyphosphoryl)methyl)pentanedioate(1.70 g, 3.438 mmol) in anhydrous acetonitrile (40 ml) Me₃SiBr (3.6 ml,27.5 mmol) was added at 0° C. The mixture was kept at 0° C. overnight,then evaporated and codistilled with MeCN, dissolved in dioxane (30 ml)and treated with water (240 □l, 13.3 mmol). The solution was stirred 30min at room temperature then toluen (30 ml) was added and the solventswere evaporated. To a solution of resulting phosphonic acid,triphenylphosphine (5.4 g, 20.6 mmol) and4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (2.68 g, 20.6 mmol) in THF(80 ml) DIAD (4.0 ml, 20.6 mmol) was added dropwise. The mixture wasstirred 6 h at room temperature. The solvent was removed in vacuo andthe residue was chromatographed on a silica gel column in EtOAc→3%MeOH/EtOAc. Yield 1.01 g (44%).

¹H NMR (400 MHz, CDCl₃): 0.04 (s, 9H, Si—CH₃), 0.98 (m, 2H, Si—CH₂),1.94-2.02 (m, 3H, H-3, H-1b), 2.20 (3×s, 9H, 4′—CH₃), 2.25-2.34 (m, 3H,H-1a, H-4), 2.83 (m, 1H, H-2), 4.16 (m, 2H, OCH₂(TMSE)), 4.77-4.93 (m,6H, 3′—CH₂).

¹³C NMR (101 MHz, CDCl₃): −1.56 (Si—CH₃), 9.30, 9.33 (4″—CH₃), 17.24(Si—CH₂), 28.15 (d, J_(1-P)=141.5, C-1), 28.26 (d, J_(3-P)=14.5, C-3),31.16 (C-4), 38.76 (d, J_(2-P)=3.8, C-2), 54.37 (COO—CH₂-3′), 55.23,55.35 (2×d, J_(C-O-P)=5.8, 6.0, P—O—CH₂), 63.02 (OCH₂ (TMSE)), 132.96(C-3″), 133.29, 133.30 (2×d, J_(3″-P)=6.0, C-3′), 140.30, 140.31, 140.44(C-4″), 151.71, 151.74, 152.07 (C-1″), 172.24 (C-5), 172.93 (d,J_(C-2-1-P)=6.6, COO).

ESI MS: 685 ([M+Na]⁺).

HR ESI MS: calcd for C₂₆H₃₅O₁₆NaPSi 685.13242; found 685.13262.

4-((hydroxy((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)-5-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-5-oxopentanoicacid TT-200714

A solution of 1-((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)5-(2-(trimethylsilyl)ethyl)2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pentanedioate (690 mg, 1.04 mmol) in anhydrous dichloromethane (10 ml)was treated with TFA (1 ml) at 0° C. The mixture was stirred 48 h at 0°C. then evaporated and the residue was chromatographed twice on a silicagel column in EtOAc→50% MeOH/EtOAc. Yield 110 mg (23%).

¹H NMR (400 MHz, CD3COCD₃): 1.80-2.12 (m, 4H, H-3, H-1), 2.21 (bs, 6H,2×CH₃), 2.32, 2.40 (2×bs, 2H, H-4), 2.90 (bs, 1H, H-2), 4.74-5.09 (m,4H, O—CH₂).

¹³C NMR (101 MHz, CD3COCD₃): 9.34, 9.43 (2×CH₃), 29.0 (C-1, C-3), 31.7(C-4), 40.1 (C-2), 54.92, 55.22 (2×OCH₂), 134.69, 136.26 (C-3′), 139.88,141.21 (C-4′), 152.97, 153.22 (C-1″), 172.15 (C-5), 175.50 (COO).

ESI MS: 449 ([M−H]⁻).

HR ESI MS: calcd for C₁₆H₁₈O₁₃P 449.04905; found 449.04895.

Bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)₂-((diethoxyphosphoryl)methyl)pentanedioate TT-301014

Dibenzyl 2-((diethoxyphosphoryl)methyl)pentanedioate (2.31 g, 5.0 mmol)was hydrogenated in anhydrous THF (100 ml) in the presence of catalyticamount of 10% Pd/C at room temperature and 1 atm overnight. The catalystwas filtered off and 4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (1.49g, 11.0 mmol), HOBt (1.49 g, 11.0 mmol) were added. A solution ofEDC.HCl (2.11 g, 11.0 mmol) and DMAP (1.34 g, 11.0 mmol) in DMF (60 ml)was added and the mixture was stirred at room temperature overnight.Solvents were evaporated and the residue was partitioned betweenEtOAc—H₂O (1:1, 200 ml). The aqueous solution was extracted with EtOAc(100 ml) again and combined organic extracts were dried (MgSO₄) andevaporated. The residue was chromatographed on a silica gel column inEtOAc→2% MeOH/EtOAc. Yield 2.13 g (84%) of oil.

¹H NMR (400 MHz, CDCl₃): 1.30-1.33 (m, 6H, CH₃(Et)), 1.87 (ddd, 1H,J_(1b-P)=18.6, J_(gem)=15.4, Jib-2=5.5, H-1b), 1.96-2.08 (m, 2H, H-3),2.18, 2.19 (2×s, 2×3H, 4′—CH₃), 2.21 (ddd, J_(1a-P)=18.1, J_(gem)=15.4,J_(1a-2)=8.5, H-1a), 2.32-2.44 (m, 2H, H-4), 2.84 (m, 1H, H-2),4.05-4.13 (m, 4H, CH₂ (Et)), 4.82-4.93 (m, 4H, 3′—CH₂). ¹³C NMR (101MHz, CDCl₃): 9.35, 9.37 (4′—CH₃), 16.36, 16.37 (2×d, J_(CH3-P)=5.9,CH₃(Et)), 27.72 (d, =143.2, C-1), 27.86 (d, J_(3-P)=12.6, C-3), 30.88(C-4), 38.96 (d, J_(2-P)=3.5, C-2), 53.84, 54.22 (3′—CH₂), 61.91-62.00(m, CH₂ (Et)), 133.15, 133.30 (C-3′), 140.17, 140.30 (C-4″), 152.01,152.05 (C-1″), 171.72 (C-5), 173.29 (d, J_(C-2-1-P)=8.6, COO).

ESI MS: 529 ([M+Na]⁺).

HR ESI MS: calcd for C₂₀H₂₇O₁₃NaP 529.10815; found 529.10819.

(5-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)-2-(((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)-5-oxopentyl)phosphonicacid TT-120814

A solution of bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)2-((diethoxyphosphoryl)methyl) pentanedioate (253 mg, 0.5 mmol) inanhydrous acetonitrile (5 ml) was treated with Me₃SiBr (530 μl, 4.0mmol) at 0° C. The solution was kept at 0° C. overnight. The volatileswere evaporated and the residue was codistilled with MeCN and treatedwith water (0.25 ml). The sample was purified on a C-18 reverse phasecolumn in gradient H₂O 55% MeOH/H₂O. Yield 170 mg (75%).

ESI MS: 449 ([M−H]⁻).

HR ESI MS: calcd for C₁₆H₁₈O₁₃P 449.04905; found 449.04877.

Bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pentanedioateTT-190215

A solution of bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)2-((diethoxyphosphoryl)methyl) pentanedioate (250 mg, 0.494 mmol) inanhydrous acetonitrile (5 ml) was treated with Me₃SiBr (530 μl, 4.0mmol) at 0° C. The solution was kept at 0° C. overnight. The volatileswere evaporated and the residue was codistilled with MeCN, dissolved indioxane (5 ml) and treated with water (36 μl, 2.0 mmol). The solutionwas stirred 30 min at room temperature then toluen (5 ml) was added andthe solvents were evaporated. To a solution of resulting phosphonicacid, triphenylphosphine (777 mg, 2.96 mmol) and4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (386 mg, 2.96 mmol) in THF(10 ml, DIAD (583 μl, 2.96 mmol) was added dropwise. The mixture wasstirred 6 h at room temperature. The solvent was removed in vacuo andthe residue was chromatographed on a silica gel column in EtOAc→4%MeOH/EtOAc. Yield 160 mg (48%).

¹H NMR (400 MHz, CDCl₃): 1.93-2.01 (m, 3H, H-3, H-1b), 2.19, 2.20 (3×s,3H, 6H, 3H, 4×CH₃), 2.24-2.45 (m, 3H, H-4, H-1a), 2.82 (m, 1H, H-2),4.77-4.96 (m, 8H, O—CH₂).

¹³C NMR (101 MHz, CDCl₃): 9.24, 9.27, 9.29 (4×CH₃), 27.90 (d, J3-P=14.0,C-3), 28.07 (d, J1-P=141.8, C-1), 30.67 (C-4), 38.51 (d, J2-P=3.7, C-2),53.88, 54.40 (2×OCH₂), 55.23, 55.37 (2×d, JCH2-O—P=6.0 and 5.8,CH₂—O—P), 132.89-133.30 (m, C-3′), 140.23, 140.34, 140.51 (C-4′),151.72, 151.75, 152.05, 152.07 (C-1″), 171.49 (C-5), 172.74 (d,JC-P=7.4, COO).

ESI MS: 697 ([M+Na]⁺).

HR ESI MS: calcd for C₂₆H₂₇O₁₉NaP 697.07764; found 697.07752.

Bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)2-((hydroxy((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pentanedioateTT-110814

A solution of bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methyl)2-((bis((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)phosphoryl)methyl)pentanedioate (256 mg, 0.38 mmol) and LiN3 (37 mg,1.07 mmol) in DMF (2 ml) was stirred at room temperature overnight. Thesolvent was evaporated and the residue was purified on a silica gelcolumn in 10-50% MeOH/EtOAc. Yield 136 mg (64%).

¹H NMR (400 MHz, CDCl₃): 1.74-2.07 (m, 4H, H-3, H-1), 2.16, 2.18, 2.18(3×s, 9H, 3×CH₃), 2.26-2.41 (m, 2H, H-4), 2.78 (m, 1H, H-2), 4.69-4.96(m, 6H, O—CH₂).

¹³C NMR (101 MHz, CDCl₃): 9.02, 9.20, 9.22 (3×CH₃), 27.80-29.13 (C-1,C-3), 30.78 (C-4), 39.62 (C-2), 53.92, 54.28, 54.44 (3×OCH₂), 133.23,133.33 (C-3′), 134.94 (d, J_(3′-C-O-P)=7.0, C-3′), 139.23, 140.39,140.48 (C-4′), 152.22, 152.29, 152.46 (C-1″), 172.57 (C-5), 174.78 (m,COO).

ESI MS: 561 ([M−H]⁻)

HR ESI MS: calcd for C₂₁H₂₂O₁₆P 561.06509; found 561.06496.

Example 3 Selective Deprotection of Phosphonate Diesters

Bromotrimethylsilane (9.3 mL; 70 mmol) was added at 0° C. to a solutionof appropriate phosphonate diester (17.5 mmol) in acetonitrile (100 mL)and kept at 0° C. for 24 h. The solution was evaporated, the residuecoevaporated with acetonitrile, followed by water and toluene. The crudeproduct was purified by chromatography on silica gel in systemchloroform-ethyl acetate-methanol (2:2:1).

The following compounds were prepared:

[5-(Methyloxy)-2-(methoxycarbonyl)-5-oxopentyl]phosphonic Acid.TT-140113

Yield: 3.2 g (73%) of a colorless syrup. ¹H NMR (CDCl₃, ppm) δ: 1.84 (m,1H, H-1a); 1.97 (m, 2H, H-3); 2.15 (m, 1H, H-1 b); 2.36 (m, 2H, H-4);2.79 (m, 1H, H-2); 3.66 (s, 3H, R₁=Me); 3.69 (s, 3H, R₂=Me). ¹³C NMR(CDCl₃, ppm) δ:29.53 (d, J_(C,P)=12.8, C-3); 30.33 (d, J_(C,P)=140.4,C-1); 32.10 (C-4); 40.89 (d, J_(C,P)=3.4, C-2); 52.43 (R₁=Me); 52.47(R₂=Me); 174.83 (COOR₁); 176.44 (d, J_(C,P)=8.1, COOR₂). ESI MS, m/z:

[5-(Benzyloxy)-2-(methoxycarbonyl)-5-oxopentyl]phosphonic Acid.TT-100313

Yield: 4.5 g (78%) of a colorless syrup. The crude product was used forthe preparation of compound TT-110313 without purification andidentification.

[5-(Benzyloxy)-2-(ethoxycarbonyl)-5-oxopentyl]phosphonic Acid. MK-800

Yield: 4.0 g (66%) of a white waxy solid. ESI MS, m/z: 687.3 [2M−H]⁻(43), 343.1 [M−H]⁻ (50). HRMS (ESI): For C₁₅H₂₀O₇P [M−H]⁻ calculated:343.09521; found: 34309513.

[5-Ethoxy-2-(ethoxycarbonyl)-5-oxopentyl]phosphonic Acid. MK-797

Yield: 3.0 g (60%) of a colorless syrup. ³¹P{¹H} NMR (CDCl₃, ppm) δ:26.70. NMR (CDCl₃, ppm) δ: 1.29 (m, 6H, 2×CH₃, Et), 2.06 (m, 3H), 2.27(m, 3H), 2.83 (m, 1H, H−2), 4.14 (m, 4H, 2×CH₂, Et). ESI MS, m/z: 563.3[2M−H]⁻ (100), 281.2 [M−H]⁻ (62). HRMS (ESI): For C₁₀H₁₈O₇P [M−H]⁻calculated: 281.07956; found: 281.07958. Anal. Calcd. for C₁₀H₁₉O₇P: C,42.56; H, 6.79; P, 10.97. Found: C, 42.07; H, 6.87; P, 10.59.

[5-Oxo-5-propoxy-2-(propoxycarbonyl)pentyl]phosphonic Acid. MK-801

Yield: 3.6 g (67%) of a white waxy solid. ³¹P {¹H} NMR (CD₃OD, ppm) δ:21.38. ¹H NMR (CD₃OD, ppm) δ: 0.92 (m, 6H, 2×CH₃, Pr), 1.39 (m, 2H),1.67 (m, 3H), 2.09 (m, 3H), 2.31 (m, 2H), 2.88 (m, 1H), 4.16 (m, 4H).ESI MS, m/z: 619.3 [2M−H]⁻ (100), 309.1 [M−H]⁻ (47). HRMS (ESI): ForC₁₂H₂₂O₇P [M−H]⁻ calculated: 309.11086; found: 309.11101.

[5-(Benzyloxy)-2-(benzyloxycarbonyl)-5-oxopentyl]phosphonic Acid. MK-824

Yield: 6.0 g (85%) of a colorless syrup. ESI MS, m/z: 811.3 [2M−H]⁻(12), 405.1 [M−H]⁻ (26).

Example 4 Esterification of Phosphonic Acids—POM Esters, POC Esters,Alkyl Esters

DBU (2 mmol) was added to a solution of appropriate phosphonic acid (1mmol) in dry dioxane (10 mL) and then heated with POC—Cl (20 mmol, 80°C., 4 h) or POM-C₁ (4 mmol, reflux, 6 h) or decyl bromide (2.3 mmol,reflux, 3 h) or 3,6,9,12,15,18-hexaoxaicosyl p-toluensulfonate (2.1mmol, reflux, 6 h), respectively. The reaction course was monitored byTLC in system toluene-acetone (4:1); detection was performed by sprayingof the TLC plate with a solution of phosphomolybdenic acid and heating.Reaction mixture was evaporated and the residue chromatographed on asilica gel column (200 mL) in system toluene-acetone (4:1) for POM andPOC esters, toluene-acetone (8:1) for decyl ester, or 12% MelH/CHCl₃ for3,6,9,12,15,18-hexaoxaicosyl ester.

The following compounds were prepared:

5-Benzyl 1-methyl2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-pentanedioate.MK-792

Yield: 194 mg (34%) of a colourless syrup. ³¹P{¹H} NMR (CDCl₃, ppm) δ:29.85. ¹H NMR (CDCl₃, ppm) δ: 1.31-1.33 (m, 12H, CH₃ iPr), 1.96-2.06 (m,3H, H-3, H-1b), 2.34-2.44 (m, 3H, H-4, H-1a), 2.84 (m, 1H, H-2), 3.69(s, 3H, COOCH₃ ), 4.93 (2× septet, 2H, J_(CH,CH3)=6.3, 2× CH iPr), 5.11(s, 2H, CH₂Bn), 5.60-5.68 (m, 4H, 2× OCH₂O), 7.30-7.38 (m, 5H,H-2′,3′,4′). ¹³C NMR (CDCl₃, ppm) δ: 21.59 (CH₃ iPr), 28.16 (d,J_(3,P)=13.6, C-3), 28.54 (d, J_(1,P)=143.2, C-1), 31.34 (C-4), 38.65(d, J_(2,P)=3.6, C-2), 52.15 (OCH₃), 66.41 (CH₂Bn), 73.27 (CH iPr),83.94 and 83.95 (2× d, J_(C-O-P)=6.2, OCH₂O), 128.25 (C-2′), 128.26(C-4′), 128.54 (C-3′), 135.76 (C-1′), 153.11 and 153.13 (OC(O)O), 172.12(C-5), 173.82 (d, J_(C-C-C-P)=8.7, 2-COO). ESI MS, m/z: 585.5 [M+Na]⁺(100), 563.5 [MH]⁺ (31). HRMS (ESI): For C₂₄H₃₆O₁₃P [MH]⁺ calculated:563.18880; found: 563.18868.

5-Benzyl 1-methyl 2-((bis(decyloxy)phosphoryl)methyl)pentanedioate.MK-796

Yield: 351 mg (57%) of a colourless syrup. ³¹P{¹H} NMR (CDCl₃, ppm) δ:28.39. ¹H NMR (CDCl₃, ppm) δ: 0.90 (7, 6H, J_(CH3,CH2)=6.9, 2× CH₃,decyl), 1.30 (m, 28H, 14× CH₂, decyl), 1.65 (m, 4H, CH₂), 1.86 (ddd,1H), 2.03 (m, 2H), 2.26 (ddd, 1H), 2.41 (m, 2H), 2.83 (tdd, 1H, H-2),3.71 (s, 3H, COOCH₃), 4.00 (qd, 4H, 2×OCH₂, decyl), 5.13 (s, 2H, CH₂,Bn), 7.36 (m, 5H, H-arom.). ESI MS, m/z: 1243.8 [2M+Na]⁺ (38), 633.4[M+Na]⁺ (100). HRMS (ESI): For C₃₄H₅₉O₇PNa [M+Na]⁺ calculated:633.38906; found: 633.38806.

Diethyl 2-({bis[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate.MK-798

Yield: 429 mg (84%) of a yellowish syrup. ³¹P{′H} NMR (CDCl₃, ppm) δ:29.46. ¹H NMR (CDCl₃, ppm) δ: 1.25 (m, 24H, 8× CH₃, POM, Et), 1.99 (m,3H), 2.34 (m, 3H), 2.80 (m, 1H, H-2), 4.17 (m, 4H, 2× CH₂, Et), 5.67 (m,4H, 2×OCH₂O). ESI MS, m/z: 533.1 [M+Na]⁺ (100). HRMS (ESI): ForC₂₂H₃₉O₁₁PNa [M+Na]⁺ calculated: 533.21222; found: 533.21221. Anal.Calcd. for C₂₂H₃₉₀₁₁P: C, 51.76; H, 7.70; P, 6.07. Found: C, 51.98; H,7.55; P, 6.01.

5-Benzyl 1-ethyl2-({bis[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate. MK-803

Yield: 183 mg (32%) of a colourless syrup. ³¹P{′H} NMR (CDCl₃, ppm) δ:29.40. ¹H NMR (CDCl₃, ppm) δ: 1.25 (m, 21H, 7× CH₃, POM, Et), 2.01 (m,3H), 2.39 (m, 3H), 2.82 (m, 1H, H-2), 4.17 (q, 2H, J_(CH2,CH3)=7.1, CH₂,Et), 5.13 (s, 2H, CH₂, Bn), 5.66 (d, 2H, J=13.1, OCH₂O), 5.67 (d, 2H,J=13.1, OCH₂O), 7.36 (m, 5H, H-arom.). ESI MS, m/z: 595.3 [M+Na]⁺ (100).HRMS (ESI): For C₂₇H₄₁O₁₁PNa [M+Na]⁺ calculated: 595.22787; found:595.22783.

5-Benzyl 1-ethyl2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-pentanedioate.MK-805

Yield: 217 mg (38%) of a colourless syrup. ³¹P{¹H} NMR (CDCl₃, ppm) δ:29.46. ¹H NMR (CDCl₃, ppm) δ: 1.27 (t, 3H, J_(CH3,CH2)=7.1, CH₃, Et),1.33 (d, 6H, J_(CH3,CH)=1.6, 2× CH₃, iPr), 1.34 (d, 6H, J_(CH3,CH)=1.6,2× CH₃, iPr), 2.04 (m, 3H), 2.41 (m, 3H), 2.83 (m, 1H, H-2), 4.17 (q,2H, J_(CH2,CH3)=7.1, CH₂, Et), 5.13 (s, 2H, CH₂, Bn), 5.66 (m, 4H,2×OCH₂O, POC), 7.36 (m, 5H, H-arom.). ESI MS, m/z: 1175.7 [2M+Na]⁺ (6),599.3 [M+Na]⁺ (100). HRMS (ESI): For C₂₅H₃₈O₁₃PNa [M+Na]⁺ calculated:577.20445; found: 577.20468.

Dibenzyl2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)pentanedioate.MK-825

Yield: 247 mg (39%) of a colourless syrup. ³¹P{¹H} NMR (CDCl₃, ppm) δ:29.31. ¹H NMR (CDCl₃, ppm) δ: 1.34 (4×d, 12H, 4×CH₃, POC), 2.04 (m, 3H),2.37 (m, 3H), 2.90 (m, 1H, H-2), 4.93 (m, 21H, 2× CH, POC), 5.11 (s, 2H,CH₂, Bn), 5.14 (d, 2H, CH₂, Bn), 5.63 (m, 4H, 2×OCH₂O), 7.35 (m, 10H,H-arom.). ESI MS, m/z: 661.2 [M+Na]⁺ (100).

Dimethyl2-((bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-pentanedioate.TT-010213

Yield: 177 mg (37%) of a colourless syrup. ¹H NMR (CDCl₃, ppm) δ: 1.31(4× d, 12H, 4×Me, POM); 1.98 (m, 2H, H-3); 2.00 (ddd, 1H, J_(1a,P)=19.1,J_(gem)=15.6, J_(1a,2)=5.4, H-1a); 2.32 (m, 2H, H-4); 2.37 (ddd, 1H,J_(1b,P)=19.0, J_(gem)=15.6, J_(1a,2)=8.6, H-1b); 2.82 (m, 1H, H-2);3.65 (s, 3H, Me, R₁); 3.70 (s, 3H, Me, R₂); 4.92 (2×sept, 2H, 2×CH,POC); 5.58-5.68 (m, 4H, 2×CH₂, POC). ¹³C NMR (CDCl₃, ppm) δ: 21.58(4×CH₃, POC); 28.13 (d, J_(C,P)=13.5, C-3); 28.45 (d, J_(C,P)=143.3,C-1); 31.09 (d, J_(C,P)=1.1, C-4); 38.62 (d, J_(C,P)=3.6, C-2); 51.69(Me, R₁); 52.17 (Me, R₂); 73.28 (CH, POC); 83.91, 83.92 (2×d,J_(C,P)=6.2, 2×CH₂, POC); 153.10, 153.12 (2×CO, POC); 172.74 (COOR₁);173.83 (d, J_(C,P)=8.9, COOR₂). ESI MS, m/z:

Dimethyl 2-((bis{[(pivaloyloxy)methoxy]phosphoryl}methyl)pentanedioate.TT-201212A

Yield: 195 mg (40%) of a colourless syrup. ¹H NMR (CDCl₃, ppm) δ: 1.22(2×s, 2×9H, 6×Me, POM); 1.91-2.03 (m, 3H, H-1a+H-3); 2.26-2.38 (m, 3H,H-1b+H-4); 2.80 (dtt, 1H, J_(2,P)=13.5, J_(2,1b)=J_(2,3b)=8.4,J_(2,1a)=J_(2,3a)=5.5, H-2); 3.65 (s, 3H, Me, R₁); 3.70 (s, 3H, Me, R₂);5.61-5.67 (m, 4H, 2×CH₂, POM). ¹³C NMR (CDCl₃, ppm) δ: 26.79 (6×CH₃,POM); 28.14 (d, J_(C,P)=13.3, C-3); 28.69 (d, J_(C,P)=142.8, C-1); 31.07(d, J_(C,P)=1.1, C-4); 38.70 (C, POM); 38.71 (d, J_(C,P)=3.5, C-2);51.71 (Me, R₁); 52.17 (Me, R₂); 81.36, 81.39 (2×d, J_(C,P)=6.2, 2×CH₂,POM); 172.72 (COOR₁); 173.87 (d, J_(C,P)=9.1, COOR₂). 176.80, 176.81(2×CO, POM). ESI MS, m/z: 505.2 [M+Na]⁺ (100). HRMS (ESI): ForC₂₀H₃₅O₁₁PNa [M+Na]⁺ calculated: 505.18092; found: 505.18099.

Dimethyl2-((bis((3,6,9,12,15,18-hexaoxaicosyl)oxy)phosphoryl)methyl)pentanedioateTT-250113

Yield: 342 mg (41%) of a colourless syrup. ¹H NMR (CDCl₃, ppm) δ: 1.20(t, 6H, J_(14′,13′)=7.0, H-14′); 1.94 (ddd, 1H, J_(1a,P)=18.8,J_(gem)=15.5, J_(1a,2)=5.6, H-1a); 1.98 (m, 2H, H-3); 2.28 (ddd, 1H,J_(1b,P)=18.5, J_(gem)=15.5, J_(1b,2)=8.4, H-1b); 2.33 (m, 2H, H-4);2.82 (m, 1H, H-2); 3.51 (q, 4H, J_(13′,14′)=7.0, H-13′); 3.56-3.68 (m,44H, H-2′ to H-12′); 3.65 (s, 3H, Me, R₁); 3.69 (s, 3H, Me, R₂); 4.15(m, 4H, H-1′). ¹³C NMR (CDCl₃, ppm) δ: 15.11 (C-14′); 27.65 (d,J_(C,P)=143.3, C-1); 28.17 (d, J_(C,P)=12.6, C-3); 31.16 (d,J_(C,P)=1.0, C-4); 38.99 (d, J_(C,P)=3.6, C-2); 51.66 (Me, R₁); 52.04(Me, R₂); 64.71, 64.76 (2×d, J_(C,P)=5.9, 2×C-1′); 66.60 (C-13′);70.60-69.77 (m, C-2′ to C-12′); 170.90 (COOR₁); 174.32 (d, J_(C,P)=9.2,COOR₂). ESI MS, m/z: 861.5 [M+Na]⁺ (100). FIRMS (ESI): For C₃₆H₇₂O₁₉P[M+H]⁺ calculated: 839.43999; found: 839.44023.

Example 5 Esterification of Phosphonic Acids-Alkyl Salicylyl Esters

Catalytic amount of DMF (10 μL), followed by oxalyl chloride (0.6 mL; 7mmol) were added to a solution of phosphonic acid (1 mmol) in drydichloromethane (10 mL). The mixture was stirred for 2 h and evaporated.The residue (intermediary phosphochloridate) was dissolved indichloromethane (5 mL), cooled to −10° C. and dry pyridine (0.16 mL) wasadded dropwise. The resulting mixture was immediately added to a cooled(−30° C.) mixture of butyl salicylate (0.4 g; 2.1 mmol) and triethylamine (0.85 mL) in dichloromethane (8 mL). The reaction mixture waswarmed slowly to room temperature, then stirred for 12 h and evaporated.The residue was chromatographed on a column of silica gel (80 mL) insystem toluene-acetone (10:1). The following compounds were prepared:

5-Benzyl 1-methyl2-({bis[2-(butoxycarbonyl)phenoxy]phosphoryl}methyl)pentane-dioate.MK-794

Yield: 0.41 g (60%) of a yellowish syrup. ³¹P{¹H} NMR (CDCl₃, ppm) δ:23.45. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 0.965 (t, 3H, J_(CH3,CH2)=7.4,CH₃), 0.968 (t, 3H, J_(CH3,CH2)=7.4, CH₃), 1.46 (m, 4H, CH₂), 1.73 (m,4H, CH₂), 2.18 (m, 2H), 2.51 (m, 3H), 2.80 (m, 1H), 3.18 (m, 1H), 3.66(s, 3H, COOCH₃ ), 4.29 (2× t, 4H, J_(CH2,CH2)=6.7, OCH₂ (Bu)), 5.11 (s,2H, CH₂(Bn)), 7.23 (m, 4H, H-arom.), 7.34 (m, 5H, H-arom.), 7.40 (m, 2H,H-arom.), 7.88 (m, 2H, H-arom.). ESI MS, m/z: 1387.3 [2M+Na]⁺ (12),705.1 [M+Na]⁺ (100), 683.1 [MH]⁺ (48). HRMS (ESI): For C₃₆H₄₃O₁₁PNa[M+Na]⁺ calculated: 705.24352; found: 705.24280.

Dimethyl2-({bis[2-(butoxycarbonyl)phenoxy]phosphoryl}methyl)pentanedioate.TT-100113

Yield: 0.48 g (79%) of a yellowish syrup. NMR (CDCl₃, ppm) δ: 0.95 (2×t,6H, J_(4″,3″)=7.4, H-4″); 1.43 (m, 4H, H-3″); 1.68-1.74 (m, 4H, H-2″);2.13 (m, 2H, H-3); 2.41 (m, 2H, H-4); 2.50 (ddd, 1H, J_(1a,P)=19.8,J_(gem)=15.5, J_(1a,2)=6.1, H-1a); 2.79 (ddd, 1H, J_(1b,P)=19.4,J_(gem)=15.5, J_(1b,2)=7.7, H-1b); 3.18 (m, 1H, H-2); 3.64 (s, 3H, Me,R₁); 3.65 (s, 3H, Me, R₂); 4.28 (2×t, 4H, J_(1″,2″)=6.8, H-1″); 7.19 (m,2H, H-5′); 7.21 (m, 2H, H-3′); 7.39 (m, 2H, H-4′); 7.86 (m, 2H, H-6′).¹³C NMR (CDCl₃, ppm) S: 13.70 (C-4″); 19.18 (C-3″); 28.17 (d,J_(C,P)=12.4, C-3); 28.37 (d, J_(C,P)=144.7, C-1); 30.63 (C-2″); 31.24(d, J_(C,P)=0.8, C-4); 38.76 (d, J_(C,P)=3.4, C-2); 51.59 (Me, R₁);52.04 (Me, R₂); 65.03, 65.05 (C-1″); 122.49, 122.54 (2×d, J_(C,P)0.9,C-3′); 123.36, 123.40 (C-1′); 124.90, 124.91 (2× d, J_(C,P)=1.4, C-5′);133.52, 133.55 (2×d, J_(C,P)=0.6, C-6′); 133.34, 133.35 (C-4′); 149.12,149.13 (2×d, J_(C,P)=9.4, C-2′); 164.64, 164.67 (2×d, J_(C,P)=1.1;CO₀Bu); 172.99 (COOR₁); 174.20 (d, J_(C,P)=11.0, COOR₂). ESI MS, m/z:629.3 [M+Na]⁺ (100). HRMS (ESI): For C₃₀H₃₉O₁₁PNa [M+Na]⁺ calculated:629.21222; found: 629.21169.

Example 6 Synthesis of Bis Amidates

Catalytic amount of DMF (10 μL), followed by oxalyl chloride (0.6 mL; 7mmol) were added to a solution of phosphonic acid (1 mmol) in drydichloromethane (10 mL). The mixture was stirred for 2 h and evaporated.The residue (intermediary phosphochloridate) was dissolved indichloromethane (5 mL), cooled to −10° C. and dry pyridine (0.16 mL) wasadded dropwise. The resulting mixture was immediately added to a cooled(−30° C.) mixture of 1-phenylalanine ethyl ester hydrochloride (0.48 g;2.1 mmol) and triethyl amine (0.85 mL) in dichloromethane (8 mL). Thereaction mixture was warmed slowly to room temperature, then stirredovernight and finally washed with an aqueous solution of citric acid (20ml). Organic portion was dried and concentrated. The residue waschromatographed on a column of silica gel (80 mL) in systemMeOH-EtOAc-CHCl₃ (4:50:50).

The following compound was prepared:

Dimethyl2-((bis(((S)-1-ethoxy-1-oxo-3-phenylpropan-2-yl)amino)phosphoryl)methyl)pentanedioateTT-280113

Yield: 0.39 g (48%) of a yellowish syrup (mixture of diastereomers). ¹HNMR (CDCl₃, ppm) δ: 1.20, 1.21, 1.23 (4×t, 6H, J_(2″,1″)=7.1, H-2″);1.19, 1.35 (2×m, 1H, H-1a); 1.73-1.98 (m, 3H, H-1 b+H-3); 2.13-2.32 (m,2H, H-4); 2.59, 2.73 (m, 1H, H-2); 2.77, 2.85, 2.95, 3.07 (4×m, 4H,H-3′); 3.58, 3.61 (2×s, 3H, Me, R₂); 3.65, 3.66 (2×s, 3H, Me, R₁); 3.95,4.04 (2×m, 2H, H-1″); 4.07-4.28 (m, 4H, H-2′+H-1″); 7.10, 7.18 (2×m, 4H,H-5′); 7.20-7.32 (m, 6H, H-6′+H-7′). ¹³C NMR (CDCl₃, ppm) δ: 14.04,14.05, 14.08 (C-2″); 28.69, 28.81 (2×d, J_(C,P)=14.0 and 15.0, C-3);30.91 (2×d, J_(C,P)=115.9, C-1); 31.01 (C-4); 39.11, 39.25 (2×d,J_(C,P)=3.3 and 3.6, C-2); 40.30, 40.46, 40.81, 40.93 (4×d, J_(C,P)=4.4,4.5, 4.9 and 5.4, C-3′); 51.66, 51.67 (Me, R₁); 52.07, 52.13 (Me, R₂);53.56, 53.94, 53.96, 54.37 (C-2′); 61.21, 61.22, 61.25, 61.33 (C-1″);126.82, 126.87, 126.90, 126.91 (C-7′); 128.36, 128.41, 128.42, 128.44(C-6′); 129.47, 129.59, 129.65, 129.69 (C-5′); 136.30, 136.37, 136.64,136.67 (C-4′); 172.95, 172.98, 173.00, 173.02 (COOR₁); 173.16, 173.22(2×d, J_(C,P)=3.1 and 2.4, C-1′); 175.03, 175.45 (2×d, J_(C,P)=4.8 and4.1, COOR₂). ESI MS, m/z: 626.9 [M+Na]⁺ (100). HRMS (ESI): ForC₃₀H₄₁O₉N₂PNa [M+Na]⁺ calculated: 627.24419; found: 627.24396.

Example 7 Synthesis of Monoesters

10% Pd/C (90 mg) was added to a solution of benzyl ester (1 mmol) in THF(30 mL) and the mixture was hydrogenated at room temperature andatmospheric pressure for 15 h. The catalyst was removed by filtrationthrough a pad of Celite. The crude filtrate was finally purified byadditional filtration through a Whatman nylon membrane filter. Thefiltrate was evaporated to give a desired monoester as a colorless syrupin yield 90-100%. The reaction course was monitored by TLC in systemethyl acetate-acetone-ethanol-water (18:3:2:2), detection was performedby spraying with bromocresol green solution and heating (white spot ofthe product).

The following products were prepared:

4-({Bis[(pivaloyloxy)methoxy]phosphoryl}methyl)-5-methoxy-5-oxopentanoicAcid. TT 150313

¹H NMR (400 MHz, CDCl₃, ppm) δ: 1.25 (s, 18H, 6×CH₃), 2.02 (m, 3H), 2.38(m, 3H), 2.85 (m, 1H, H-2), 3.73 (s, 3H, COOCH₃ ), 5.67 (d, 4H, J=13.1,2×OCH₂O).

4-((Bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-5-methoxy-5-oxopentanoicAcid. MK-793

³¹P{¹H} NMR (CDCl₃, ppm) δ: 30.01. ¹H NMR (400 MHz, CDCl₃, ppm) δ:1.32-1.34 (m, 12H, CH₃), 1.98-2.08 (m, 3H, H-3, H-1b), 2.33-2.44 (m, 3H,H-4, H-1a), 2.86 (m, 1H, H-2), 3.72 (s, 3H, COOCH₃ ), 4.94 (2×sept, 2H,OCH(CH₃)₂), 5.61-5.69 (m, 4H, OCH₂O). ¹³C NMR (CDCl₃, ppm) δ: 21.59(CH₃), 27.85 (d, J3, p=12.9, C-3), 28.40 (d, J1, P=143.3, C-1), 30.94(C-4), 38.51 (d, J_(2,P)=3.6, C-2), 52.21 (OCH₃), 73.52 (CH iPr), 84.02and 84.03 (2× d, J_(C,P)=6.3, OCH₂O), 153.12 and 153.15 (O(CO)O), 173.83(d, J_(C,P)=9.6, COOMe), 176.38 (COOH). ESI MS, m/z: 495.4 [M+Na]⁺(100), 473.4 [MH]⁺ (58). HRMS (ESI): For C₁₇H₂₉O₁₃PNa [M+Na]⁺calculated: 495.12380; found: 495.12378.

4-({Bis[2-(butoxycarbonyl)phenoxy]phosphoryl}methyl)-5-methoxy-5-oxopentanoicAcid MK-795

³¹P{¹H} NMR (CDCl₃, ppm) δ: 24.12. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 0.95(m, 6H, CH₃), 1.44 (m, 4H, CH₂CH₃), 1.72 (m, 4H, CH₂CH₂CH₃), 2.05-2.19(m, 2H, H-3), 2.39-2.57 (m, 3H, H-1a, H-4), 2.82 (ddd, 1H,J_(1b,P)=19.6, J_(gem)15.6, J_(1b,2)=7.6, H-1b), 3.20 (m, 1H, H-2), 3.66(s, 3H, OCH₃ ), 4.27-4.31 (m, 4H, COOCH₂ ), 7.19-7.25 (m, 4H, H-arom.),7.40 (m, 2H, H-arom.), 7.87 (m, 2H, H-arom.). ¹³C NMR (CDCl₃, ppm) δ:13.61 (CH₃), 19.11 (CH₂ CH₃), 27.85 (d, J_(3,P)=11.9, C-3), 28.28 (d,J_(1,P)=144.8, C-1), 30.58 (CH₂ CH₂CH₃), 31.12 (C-4), 38.62 (d,J_(2,P)=3.1, C-2), 51.96 (OCH₃), 65.01 and 65.02 (2×OCH₂), 122.43-122.50(m, C-3′), 123.31-123.37 (m, C-1′), 124.91 (C-5′), 131.48 and 131.50(C-6′), 133.32 (C-4′), 149.01-149.10 (m, C-2′), 164.59 and 164.62(COOBu), 174.08 (d, J_(C,P)=11.5, COOMe), 177.32 (COOH). ESI MS, m/z:615.2 [M+Na]⁺ (100). HRMS (ESI): For C₂₉H₃₇O₁₁PNa [M+Na]⁺ calculated:615.19657; found: 615.19577.

4-{[Bis(decyloxy)phosphoryl]methyl}-5-methoxy-5-oxopentanoic Acid.MK-799

³¹P{¹H} NMR (CDCl₃, ppm) δ: 29.45. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 0.88(t, 6H, J_(CH3,CH2)=7.1, CH₃ (H-10′)), 1.23-1.36 (m, 28H, CH₂(H-3′-H-9′)), 1.61-1.67 (m, 4H, CH₂ (H-2′)), 1.90 (ddd, 1H,J_(1a,P)=18.5, J_(gem)=15.5, J_(1a,2)=5.5, H-1a), 1.95-2.05 (m, 2H,H-3), 2.26 (ddd, 1H, J_(1b,P)=18.3, J_(gem)=15.5, J_(1b,2)=8.2, H-1b),2.31-2.43 (m, 2H, H-4), 2.84 (m, 1H, H-2), 3.70 (s, 3H, COOCH₃ ),3.98-4.02 (m, 4H, CH₂ (H-1′)). ¹³C NMR (CDCl₃, ppm) δ: 14.09 (C-10′),22.65 (C-9′), 25.47 (C-3′), 27.45 (d, J_(1,P)=142.9, C-1), 28.14 (d,J_(3,P)=12.1, C-3), 29.16 (C-4′), 29.28 and 29.51 (C-5′,6′,7′), 30.46(d, J_(2,P)=6.1, C-2′), 31.12 (C-4), 31.87 (C-8′), 38.92 (d,J_(2,P)=3.5, C-2), 52.01 (OCH₃), 66.05-66.11 (m, C-1″), 174.41 (d,J_(C,P)=9.1, COOMe), 176.11 (COOH). −ESI MS, m/z: 1039.8 [2M−H]⁻ (10),519.4 [M−H]⁻ (100). HRMS (−ESI): For C₂₇H₅₂O₇P [M−H]⁻ calculated:519.34561; found: 519.34549.

4-({Bis[(pivaloyloxy)methoxy]phosphoryl}methyl)-5-ethoxy-5-oxopentanoicAcid. MK-804

³¹P{¹H} NMR (CDCl₃, ppm) δ: 29.56. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 1.25(m, 21H, CH₃), 2.01 (m, 3H), 2.37 (m, 3H), 2.83 (ddd, 1H), 4.18 (m, 2H,CH₂ CH₃), 5.68 (d, 4H, J=12.9, OCH₂O). ESI MS, m/z: 505.1 [M+Na]⁺ (100).HRMS (ESI): For C₂₀H₃₅O₁₁PNa [M+Na]⁺ calculated: 505.18092; found:505.18109.

4-((Bis{[(isopropoxycarbonyl)oxy]methoxy}phosphoryl)methyl)-5-ethoxy-5-oxopentanoicAcid. MK-806

³¹P{¹H} NMR (CDCl₃, ppm) δ: 29.72. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 1.30(m, 15H, CH₃), 2.03 (m, 3H), 2.40 (m, 3H), 2.85 (m, 1H), 4.18 (q, 2H,J_(CH2,CH3)=7.1, CH₂ CH₃), 4.94 (dq, 2H, J=12.4 and 6.2, OCH(CH₃)₂),5.61-5.75 (m, 4H, OCH₂O). ESI MS, m/z: 509.0 [M+Na]⁺ (100). HRMS (ESI):For C₁₈H₃₁O₁₃PNa [M+Na]⁺ calculated: 509.13945; found: 509.13962.

Example 8 Synthesis of Dicarboxylic Acid

Catalytic amount of DMF (10 μL), followed by oxalyl chloride (0.6 mL; 7mmol) were added to a solution of compound MK-824 (1 mmol) in drydichloromethane (10 mL). The mixture was stirred for 2 h and evaporated.The residue (intermediary phosphochloridate) was dissolved indichloromethane (5 mL), cooled to −10° C. and dry pyridine (0.16 mL) wasadded dropwise. The resulting mixture was immediately added to a cooled(−30° C.) mixture of hexadecyloxypropyl alcohol (0.63 g; 2.1 mmol) andtriethyl amine (0.85 mL) in dichloromethane (8 mL). The reaction mixturewas warmed slowly to room temperature, then stirred for 12 h andevaporated. The residue was chromatographed on a column of silica gel(80 mL) in system toluene-acetone (10:1). The fractions containingphosphonic ester intermediate were evaporated (580 mg, 60%), the residuewas hydrogenated in THF (30 mL) in the presence of 10% Pd/C (cat.) atatmospheric pressure for 24 h. The catalyst was removed by filtrationthrough a pad of celite. The crude filtrate was finally purified byadditional filtration through a Whatman nylon membrane filter. Thefiltrate was evaporated to give a desired monoester which wascrystallized from hexane in freezer (−20° C.).

The following compound was prepared:

2-((Bis(3-(hexadecyloxy)propoxy)phosphoryl)methyl)pentanedioic acid.TT-041212

Yield 670 mg (85%) of crystals. ¹H NMR (CDCl₃, ppm) δ: 0.87 (t, 6H,J_(19,18)=7.0, H-19′); 1.19-1.35 (m, 52H, H-6′ to H-18′); 1.54 (m, 4H,H-5′); 1.90 (m, 1H, H-1a); 1.91 (m, 4H, H-2′); 2.04 (m, 2H, H-3); 2.30(ddd, 1H, J_(1b,P)=18.2, J_(gem)=15.6, J_(1b,2)=8.5, H-1b); 2.47 (m, 2H,H-4); 2.83 (m, 1H, H-2); 3.39 (t, 4H, J_(4′,5′)=6.8, H-4′); 3.48 (m, 4H,H-3′); 4.12 (m, 4H, H-1′). ¹³C NMR (CDCl₃, ppm) δ: 14.11 (C-19′); 22.68(C-18′); 26.13 (C-6′); 27.24 (d, J_(C,P)=142.3, C-1); 28.04 (d,J_(C,P)=12.6, C-3); 29.70-29.36 (m, C-5′+C-7′ to C-16′); 30.64, 30.66(2×d, J_(C,P)=6.4, C-2′); 31.43 (C-4); 31.91 (C-17′); 39.11 (bd,J_(C,P)=3.3, C-2); 63.42 (d, J_(C,P)=6.5, C-1′); 66.46 (C-3′); 71.23(C-4′); 177.19 (C-5); 178.06 (d, J_(C,P)=8.4, C-6). Elem. An. forC₄₄H₈₇O₉P calc.: C 66.80, H 11.08, P 3.92; found: C 66.93, H 11.18, P3.79.

Example 9 Simple Alkyl Esters Are Not Effective 2PMPA Prodrugs

In general, the prodrugs were screened by an in vitro metabolicstability screen, and if positive, followed by an in vivo single timepoint pharmacokinetic study, and in select cases, an in vivo full timecourse pharmacokinetic study (FIG. 3). In this Example, the four acidicfunctional groups in 2-PMPA were systematically capped. The carboxylicacids were first masked with simple alkyl esters (Compounds 1, 2, and3). In vitro chemical and metabolic stability of the prodrugs then wereconducted, following 60 min incubation in plasma stability screens forprodrugs. Simple carboxylic esters like 1, 2 and 3, unexpectedly, turnedout to be too stable, likely due to a very hydrophilic nature of thephophonate containing part of their molecules (FIG. 4 shows thestability screen for compound 1).

Example 10 Capping Both Phosphonates and Alpha Carboxylate Critical forEnhancing Permeability

Masking of the phosphonate while keeping the carboxylates free as in thebis-isopropyloxycarbonyl methyl derivative (bis-POC, 4) andbis-pivaloyloxy methyl derivative (bis POM, 5) below alone is notfeasible because of the chemical instability of those derivatives. Thelikely cause is direct participation of the α-carboxylate in thehydrolysis of the POC or POM group. Combination of both approaches asillustrated by example below renders compounds 6 and 7 with goodcompound penetrability. These compounds are, however, only converted tothe corresponding carboxylate ester 1 which is stable in plasma and didnot show the ability to release 2-PMPA. Very similar results wereobtained with corresponding diethyl esters. In vitro metabolic stabilityof the 2-PMPA prodrugs 6 and 7 also were conducted. These prodrugs werefound to be stable to chemical hydrolysis and unstable in mouse plasmaand mouse liver microsomes (FIG. 5 shows the stability screen forcompound 6).

Single time point pharmacokinetic studies were then performed on the2-PMPA prodrugs to evaluate for enhancement in prodrug permeability andPMPA release. FIG. 6 shows the concentrations of 6, 7 and 8 tested andtheir comparison to 2-PMPA following oral administration at 30 mg/kgequivalent dose of 2-PMPA. Compounds 6 and 7 showed 25-50 foldenhancement in permeability when compared to 2-PMPA alone. Moreimportantly, 8 with a free γ-carboxylate also showed similar enhancementin permeability. However no release of 2-PMPA was observed from any ofthese prodrugs and thus further optimization was needed. Since it is theα-carboxylate responsible for the instability of bis-POC and bis-POMcompounds 4 and 5, derivatization of the γ-carboxylate was unnecessaryas prodrugs with free γ-carboxylate 8 and 9 also exhibited good oralavailability. But even in this case, the bioconversion only proceededmostly to monoester 10. This was also the case of corresponding ethylesters (not shown).

Example 11 Pivaloyloxymethyl (POM) and Propyloxycarbonyloxymethyl (POC)on Alpha Carboxylate Found to be Critical for Enhancement ofPermeability and Release of Free 2PMPA

In vitro metabolic stability screens of compounds JHU 2106 and 2108-2112in mouse plasma and liver subcellular fractions are shown in FIGS. 7-9and Tables 2-7. Compound JHU 2106, comprising methyl esters, was foundto be too stable and therefore hindered the release of 2-PMPA (FIG. 7A,Table 2). Compound JHU 2108 was also found to be too stable in some ofthe samples (FIG. 7B, Table 3). Compound JHU 2109 was found to fallapart easily even in HBSS buffer and therefore would not even be able toget metabolized (FIG. 8A, Table 4). Compounds JHU 2110, 2111, and 2112were found to be stable in HBSS buffer and were able to release 2-PMPAefficiently (FIGS. 8B, 9A-9B; Tables 5-7). An in vivo single time pointpharmacokinetic study (method 1) of compounds JHU 2106-2112 in micesuggested that the POM (JHU 2109) and POC (JHU 2110) prodrugs were themost permeable (FIG. 10). A more than 50-fold increase of the POM andPOC ester prodrugs/metabolites was seen following oral dosing (method 1;FIG. 7). However, the POM and POC ester prodrugs did not release 2-PMPA(method 2; FIG. 8). Increasing the ester chain length on thecarboxylates did not increase 2-PMPA release (FIGS. 9A-9B, Tables 8-9).No or minimal 2-PMPA release was observed with the ethyl and propylesters. Compounds 2236 and 2237, ethyl and alkyl ester derivatives, werefound to be too stable and not much release of 2-PMPA was observed(Tables 8-9).

However, the stability of the simple carboxylic ester could be overcomeby introducing another isopropyloxycarbonyloxy methyl or pivaloyloxymethyl moiety on the α-carboxylate. The Tris-POC (JAM0186) and Tris-POM(JAM0168) prodrugs demonstrated sufficient chemical stability,especially the POC moiety, and instability in plasma and liversubcellular fractions depicting the potential of releasing 2-PMPA (FIGS.10A-10B; scheme for synthesis of Tris-POC shown in FIG. 1). Withoutwishing to be bound to any one particular theory, it is believed thatthe double esters on the Tris-POC prodrug allow better release of theprodrug to 2-PMPA. The POM esters on the carboxylate increased 2-PMPAapproximately 18-fold following oral dosing. In vivo pharmacokineticstudies at 30 mg/kg equivalent 2-PMPA in mice showed about a 20-foldincrease in the 2-PMPA availability (FIG. 10B). This is the first timehigh micromolar concentrations of 2-PMPA have been achieved in plasmafollowing oral administration.

In addition, compound 2609, with an extra methyl group, alsodemonstrated sufficient chemical stability, and instability in plasmaand liver subcellular fractions (Table 10).

In terms of permeability of the esters on the α-carboxylate, the ethylester (compound JHU 2236) showed the most permeability, followed by themethyl ester (compound JHU 2106), and then the propyl ester (compoundJHU 2263). Even so, compounds with simple alkyl esters on theα-carboxylate, though they showed some enhancement in permeability, weretoo stable to cause the release of 2-PMPA in vivo. In addition, both themono and diesters showed comparable permeabilities and not muchdifference was observed between the mono ethyl and mono methyl esters.Except for the POM and POC esters on phosphonates (compounds JAM0168 andJAM0186), most of the other functionalities did not show a highpermeability in vivo (e.g., compounds JHU 2235 and 2238). The POM andPOC esters on phosphonate showed the highest permeability and werechosen as appropriate promoeities for phosphonic acid and furtherstructural modification were based on these for enhancement of 2-PMPApermeability and release.

TABLE 2 Stability results for JHU 2106 in different matrices. Time MouseMouse Mouse Human Human (min) HBSS Plasma S9 Microsomes PlasmaMicrosomes 0 100%  100% 100% 100% 100% 100%  30 92% 102% 101% 102% 100%90% 60 94% 101%  86% 103%  86% 96%

TABLE 3 Stability results for JHU 2108 in different matrices. Time MouseMouse Mouse (min) HBSS Plasma S9 Microsomes 0 100% 100%  100% 100% 30 99% 2% 106%  99% 60 101% 1% 100% 100%

TABLE 4 Stability results for JHU 2109 in different matrices. Time MouseMouse Mouse (min) HBSS Plasma S9 Microsomes 0 100%  100%  100%  100%  3046% 0% 1% 2% 60 21% 0% 0% 0%

TABLE 5 Stability results for JHU 2110 in different matrices. Time MouseMouse Mouse (min) HBSS Plasma S9 Microsomes 0 100% 100%  100%  100%  30101%  2%  1%  2% 60  93% 0.4% 0.1% 0.1%

TABLE 6 Stability results for JHU 2111 in different matrices. Time MouseMouse Mouse (min) HBSS Plasma S9 Microsomes 0 100% 100%  100%  100%  30100% 3% 1% 3% 60  95% 1% 2% 2%

TABLE 7 Stability results for JHU 2112 in different matrices. Time MouseMouse Mouse (min) HBSS Plasma S9 Microsomes 0 100%  100%  100%  100%  3093% 2%  0%  2% 60 93% 1% 0.1% 0.1%

TABLE 8 Stability results for JHU 2236 in different matrices. Time MouseMouse Mouse Human Human (min) Microsomes Plasma S9 Microsomes plasma 0100%  100%  100% 100%  100%  30 99% 99% 103% 88% 81% 60 88% 83% 103% 73%59%

TABLE 9 Stability results for JHU 2237 in different matrices. Time MouseMouse Mouse Human Human (min) Microsomes Plasma S9 Microsomes plasma 0100%  0% 100%  100%  100%  30 2% 0% 1% 1% 50% 60 0% 0% 0% 0% 18%

TABLE 10 Stability results for JHU 2608, 2609 and 2610 in differentspecies Time Human Human Mouse Mouse JHU# (min) Microsomes PlasmaMicrosomes Plasma JHU 0 100%  100% 100%  100%  2608 30 80% 109% 49% 0%60 61%  76% 22% 0% JHU 0 100%  100% 100%  100%  2609 30 27% 106%  1% 0%60  8%  75%  0% 0% JHU 0 100%  100% 100%  100%  2610 30 92%  98% 86% 3%60 81%  92% 68% 1%

Example 12 2-PMPA Prodrugs with Tris POC Esters in Different Species

The 2-PMPA prodrugs with the Tris-POC esters showed excellent oralbioavailability in rodents and dogs (compound JHU 2609, Table 10;compound JAM0186, FIGS. 11-14, Table 11). The Tris-POC compound enhancedexposures following oral dosing in mice and achieved more than 20-foldenhancement in permeability versus 2-PMPA (FIG. 12). The metabolicstability of the Tris-POC compound in different species was seen, withthe dog stability most similar to the human stability (FIG. 13).Relative to the mouse, the dog sample showed a 10 fold increase in theC_(max) of 2-PMPA, showing a high availability of the compound in thedog species (FIG. 14). The metabolic stability of the Tris-POC compoundcould be further enhanced in different species by the addition of amethyl group (FIG. 15).

TABLE 11 Stability results for TRIS-POC in different species Time HumanHuman Mouse Mouse (min) Microsomes Plasma Microsomes Plasma 0 100% 100%  100%  100%  30 3% 64% 1% 1% 60 0% 48% 0% 0% Time Human Human DogDog Monkey Monkey (min) Microsomes Plasma Microsomes Plasma MicrosomesPlasma 0 100%  100% 100%  100%  100%  100%  30 5% 102% 1% 67% 0% 50% 600%  77% 0% 28% 0% 11%

Example 13 Pharmacological Inhibition of PSMA as IBD Therapy

An Overview of IBD: IBD, an idiopathic, chronic and frequently disablinginflammatory disorder of the intestine, has two subtypes: Crohn'sdisease (CD) and ulcerative colitis (UC), each accounting for ˜50% ofIBD patients (Xavier and Podolsky, 2007; Strober et al., 2007; Sartor,2006). IBD is a widespread GI disease, with a prevalence of =0.2% inWestern population. In the United States alone, there are 1.4 milliondiagnosed IBD patients, resulting in enormous suffering and health-carecosts. It is increasingly clear that IBD is a complex multifactorialdisease with both genetic and environmental contributions, theinteraction of which leads to IBD (Xavier and Podolsky, 2007; Strober etal., 2007; Sartor, 2006; Kaser et al., 2010). Unfortunately, theetiology of this mucosal dysregulation in UC and CD remain elusive(Kaser et al., 2010). Despite increasing therapeutic options availablefor the management of IBD, approximately ⅓ of IBD patients do notrespond to any given therapy, and there is no cure for IBD (Hamilton etal., 2012). Anti-tumor necrosis factor (TNF)-based therapies, such asinfliximab (IFX), adalimumab and certolizumab pegol are currently themost effective therapies for severe UC and CD (Hanauer et al., 2002;Kozuch and Hanauer, 2008; Colombel et al., 2007; Schreiber et al.,2007). However, one-third of patients with CD do not respond to anti-TNFtherapies and another third lose responsiveness within six months ofinitiating therapy (Regueiro et al., 2007; Lawrance, 2014). Thesenonresponders have more aggressive mucosal immune responses andadditional treatments are indicated (Schmidt et al., 2007). Patientswith extensive disease or who are at risk for short gut syndrome due toprior resections are usually poor surgical candidates. Currently, theonly approved medication for patients who have failed an anti-TNF agentis natalizumab. However, natalizumab has been associated with severalcases of progressive and often fatal multifocal leukoencephalopathy(PML; Van et al., 2005). This emphasizes the significance of exploringand identifying new and more effective therapies in patients with IBD.

Human Validation Data: PSMA expression and enzymatic activity isselectively elevated in patient samples with IBD (FIGS. 16-17).Gene-profiling and immunohistological analyses (FIG. 16) showed thatPSMA is intensely upregulated in the intestinal mucosa of patients withCrohn's disease (Zhang et al., 2012). To further determine the relevanceof PSMA to IBD, PSMA functional enzymatic activity was examined andcompared in normal and diseased mucosa of 32 surgical intestinalspecimens from 20 subjects (FIG. 17), including healthy controls,patients with IBD, and non-IBD controls (diverticulitis), usingpreviously described methods. A 300-1,000% increase in PSMA activity wasfound in the intestinal mucosa with active IBD when compared to that inan uninvolved area of the same patients, or the intestine from healthyand non-IBD controls. These data suggest a clear positive associationbetween activation of PSMA and IBD.

Preclinical Efficacy: PSMA inhibition shows profound efficacy in threemajor animal models of IBD (FIGS. 18-21). To investigate whether PSMAcan be a suitable novel therapeutic target for clinical interventionagainst IBD, the effect of PSMA prototype inhibitors on three mostwidely used murine models of IBD was tested, including DNBS-inducedcolitis, DSS-induced colitis, and IL-10 knockout (IL-10 KO) mice (agenetic model that develops spontaneous colitis). In all three models,PSMA inhibitor treatment dramatically ameliorated symptoms. In theDNBS-induced colitis model (FIG. 18), PSMA inhibition was found to besimilar to positive control sulfasalazine. In the DSS colitis model,PSMA inhibition significantly reduced the disease activity index (FIG.19). Moreover, the PSMA activity in the colonic and cecal mucosa ofDSS-treated mice was potently inhibited by PSMA inhibitor, indicatingtarget engagement (FIG. 20). The efficacy of 2-PMPA in treatment ofspontaneous colitis in IL-10 KO mice was also remarkable. First, PSMAinhibitor 2-PMPA significantly reduced the disease severity, includingmacroscopic disease, colonic hypotrophy, and provided better stoolconsistency (FIGS. 21A-21B). More interestingly, a complete retractionof prolapse in 2 of the 20 mice (10%) treated with the inhibitor wasobserved (FIG. 21D), a phenomenon that has never been seen in more than800 IL-10 KO mice used. The improvement of these prolapse-retractingmice was unequivocally obvious in that their body weight increaseddramatically when compared to that of untreated control IL-10 KO mice(FIG. 21C). In conclusion, using three major animal models of IBD, thesignificance of PSMA as a novel therapeutic target for treatment of IBDhas been demonstrated.

Novel orally available prodrug of 2-PMPA has been identified thatexhibits >20 fold enhancement in 2-PMPA permeability in vivo: The verypotent phosphonic acid-based PSMA inhibitor termed 2-PMPA (Ki=300 pM)(Rais et al., 2014) demonstrated excellent efficacy following i.p.administration at 100 mg/kg in both the DSS as well as IL 10 knock outmodel. However, 2-PMPA is extremely hydrophilic with poor oralavailability (F<1%). Given the success of using prodrug approaches toincrease the oral bioavailability of other phosphonic acid drugs(Hepsera™ and Viread™) (Barditch-Crovo et al., 1997; Cundy et al., 1997;Barditch-Crovo et al., 2001), a similar strategy for 2-PMPA wasemployed. An orally bioavailable prodrug of 2-PMPA (Tris-POC-2-PMPA) hasbeen identified that enables ˜20 fold enhancement in permeability (FIG.17). More importantly, the prodrug afforded >10-20 fold sustainedconcentrations of liberated 2-PMPA for up to 4 hours following oraladministration.

Dose response/efficacy and pharmacokinetic studies of PSMA inhibitors(2-PMPA and its oral prodrug) in two murine models of IBD: IL-10knockout (KO) and DSS-induced colitis:

The dose response of 2-PMPA (HEPES saline as vehicle) with three doses,1, 10, and 100 mg/kg, using an i.p. delivery route, has been completedand it was observed that a dose dependent effect with 100 mg/kg providedthe most benefit. The 2-PMPA Tris POC prodrug has also been tested in apreliminary experiment at one high dose via the oral route (100 mg/kg)using a 50% PEG/water vehicle. Unfortunately, the vehicle itself showeddetrimental effects. Several FDA approved vehicles will be evaluatedincluding ethanol/tween, propylene glycol and2-Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) for solubility andcompatibility (Thackaberry, 2013). Once the optimal vehicle isidentified, a dose response of the oral 2-PMPA Tris POC prodrugdelivered at 3, 10, and 30 mg/kg p.o. (equivalent 2-PMPA) will becompleted. To evaluate efficacy, for DSS model of colitis, the prodrugwill be evaluated orally at three different doses mentioned above at thesame time (day 1) as DSS is given to induce colitis. The treatmentduration will be 7 days, as described in FIG. 23. For IL-10 KO model ofcolitis, 3 month old mice will be given the prodrug and treatmentduration will be 2 weeks, as described in FIG. 26.

For the pharmacokinetic studies, at the end of treatment on day 7 (DSSmodel) and day 14 (IL-10 KO model) at 2 hour post dose, blood andcolonic mucosa will be collected for drug PK analysis. Plasma will begenerated from blood by centrifugation and all samples will be stored at−80° C. until further analysis. Concentrations of inhibitors in plasmaand tissue will be determined via LC/MS/MS as described previously (Raiset al., 2014).

Determine the cellular and molecular mechanisms of PSMA inhibition inIBD including effects on intestinal epithelial cells, dendritic cells(DCs), and intestinal mucosal cytokine profiles: Involvement of PSMA inthe pathogenesis of IBD is novel and little is known. Explanation ofthis association is beyond the current knowledge on PSMA. Therefore, itis important to understand how PSMA is involved in IBD at the cellularand/or molecular levels.

The major site of PSMA expression in the intestine is the mucosa (FIGS.21 and 22), where immunohistological analysis shows PSMA ispredominantly expressed in the intestinal epithelial cells (FIG. 21).Therefore, to determine if the PSMA inhibitors target directly to theintestinal epithelial cells, the first logical site of drug action wouldbe the intestinal epithelial cells. The hypothesis is that in the stateof IBD, intestinal epithelial cells, which are the first defense line inthe gut to keep the commensal and invading bacteria at bay, may sense achange in luminal bacteria and respond with a surge of PSMA expression.This increase of PSMA activity would then promote subsequent secretionof proinflammatory factors such as cytokines, and trigger aninflammatory cascade that leads to IBD. To test this hypothesis, colonicepithelial cells (CECs), both Caco-2 cells (a widely used colonicepithelial cell line) and CECs isolated from mice (WT, control andIL-10KO), will be used, as schematically illustrated in FIG. 27. Fornormal CECs or Caco-2 cells, inflammatory conditions will be induced by10 nm LPS during cell culture (LPS will activate the proinflammatorycascade through TLR4 that are highly expressed on CECs). CECs isolatedfrom IL-10KO mice (3-month old) will be inflamed, and thus there is noneed for induction with LPS. Cytokine levels in culture medium(secreted) will be analyzed by multiplex ELISA (using BIORAD Bio-Plex200 System with HTF and automated washer), as previously described (Alexet al., 2009; Alex et al., 2010) for 17 different cytokines andchemokines, and those in the cells will be analyzed by RT-PCR. Thecytokines/chemokines to be analyzed include IL-1α, IL-1β, IL-2, IL-4,IL-6, IL-10, IL-12 (p40), IL-13, IL-17, IFN (Interferon)-γ, TNFα, G-CSF,CCL2 (MCP-1). CCL3 (MIP-1α), CCL4 (MIP-1β), CXCL8 (IL-8), and CXCL10(IP-10). It will be determined if LPS treatment activates the geneexpression of PSMA by directly measuring PSMA activity. If PSMAinhibitors suppress the expression and secretion of proinflammatorycytokines and/or enhance the expression and secretion ofanti-inflammatory cytokines (such as IL-10 and/or IL-22), it wouldsuggest that increased PSMA expression promotes inflammation in CECs,and thereby confirms the hypothesis that the PSMA inhibitors indeedtarget directly to the CECs.

Another target for PSMA inhibitors might be the highly specializeddendritic cells (DCs). IBD has been considered as a T-cell-driveninflammatory disease, by highly specialized immune cells calleddendritic cells (DCs). DCs determine whether T-cell responses areimmunogenic (against harmful invading pathogens) or tolerogenic (againstharmless antigens). In the gut, intestinal DCs recognize and respond tobacteria from the gut lumen and maintain intestinal immune homeostasisby generating tolerogenic T-cell responses towards the commensalmicrobiota. Previous efforts have recently demonstrated a specific DCsubset, CD103+ DCs, which can be successfully identified in the humancolon. The proportion of CD103+ DCs was reduced in patients with activeIBD (FIG. 28). Recent reports in mice demonstrate intestinal DCsexpressing the gut-homing marker m437 are required for induction ofT-reg and IL-10-producing T-cells (Villablanca et al., 2013). There isstrong evidence that expression of α₄β₇ on murine colonic DCs isconfined to the CD103+ subset. Furthermore, proportions of α₄β₇+ DCs arereduced in the inflamed colon of IL-10KO mice (FIG. 29), suggestingIL-10 may play a key role in differentiation of α₄β₇+ regulatory DCsubsets in the gut. This subset of DCs plays a critical role ofdampening the T-cell response in normal condition and is lost in theinflammatory condition in both human and murine model of colitis.

Without wishing to be bound to any one particular theory, it is thoughtthat a PSMA inhibitor may up-regulate this particular tolerogenic DCsubset, thereby reducing the T-cell-mediated inflammatory response andameliorate symptoms of colitis in IL-10 KO mice as was observed (FIG.26). Although PSMA is not normally expressed in the DCs, it is possiblethat it is expressed in these cells when under inflammatory conditions,and that PSMA expression may suppress the expansion of this specificsubset of DCs. Alternatively, it is also possible that the upregulationof PSMA in CECs activates and releases certain cellular factors thatinhibit the colonic DC expansion, resulting in the loss of thistolerogenic DCs and leading to a hyper-reactive T-cell response. To testthis hypothesis, it will first be determined whether PSMA is upregulatedin DCs in the inflammatory condition. The best model for this purpose isthe IL-10KO mice, since previous efforts have already demonstrated thetherapeutic efficacy of PSMA inhibitors and the loss of the colonictolerogenic CD103+/α₄β₇+ DC subset in IL-10KO mice. At least one of thefollowing two approaches can be employed: 1) RT-PCR: DCs can be isolatedby FACS from colonic mucosa of both WT (control) and IL-10KO mice. PSMAexpression can be analyzed by RT-PCR; 2) Immunohistology: Colonicsegments of both WT (control) and IL-10KO mice can be examined for theexpression of PSMA in DCs using CD103 and α₄β₇ as marker for DCs (triplelabeling).

To determine if PSMA inhibitors promote tolerogenic CD103+/α₄β₇+DCsubset, colonic DCs can be isolated from IL-10 KO mice that are treatedor not (control) with 2-PMPA or its prodrug, and further analyzed forCD103+/α₄β₇+ by FACS, as demonstrated in FIGS. 28-29. If the hypothesisis correct, it is expected that an increase or recovery of thetolerogenic CD103+/α₄β₇+ DC subset toward what occurs in WT mice (seeFIG. 29) in the colon of diseased IL-10KO mice will be seen.

In terms of cytokine profiling in the colonic mucosa, it is hypothesizedthat inhibition of PSMA in general may suppress the proinflammatorycytokines/chemokine and/or enhance anti-inflammatory ones in the colon.To test this hypothesis, total colonic mucosa can be isolated fromcolitis mice that are treated or not (controls), and a set of 17cytokines/chemokines (see the list above) in the total mucosal proteinextract can be analyzed by multiplex ELISA.

Summary: Recent genomic, clinical, and pharmacological data implicatethe metalloenzyme Prostate Specific Membrane Antigen (PSMA), in theetiology of inflammatory bowel disease (IBD). Data illustrate thatpharmacological inhibition of PSMA using prototype inhibitorsameliorates IBD symptoms in three preclinical models. Orally availableinhibitors have recently been synthesized and characterized. Given thesestrong findings, it is hypothesized that PSMA activates aproinflammatory signaling cascade that leads to or enhances intestinalinflammation in IBD, and that specific pharmacological inhibition willbe a novel and effective strategy for IBD therapy.

Example 14 (Prophetic) Pharmacological Inhibition of PSMA as MS Therapy

Introduction: Approximately 50% of 2.3 million Multiple Sclerosis (MS)patients worldwide experience cognitive impairment, for which there isno approved treatment (Dutta and Trapp. Neurology, 2007. 68(22 Suppl 3):p. S22-31; Calabrese, et al. Arch Neurol, 2009. 66(9): p. 1144-50),making therapies in MS cognition a large unmet medical need.N-acetylaspartylglutamate (NAAG), one of the most abundant neuropeptidesin the mammalian brain (Neale, et al. J Neurochem, 2000. 75(2): p.443-52), is thought to serve as the endogenous agonist of themetabotropic glutamate receptor 3 (mGluR3) (Olszewski, et al. SchizophrRes, 2012. 136(1-3): p. 160-1). Recent clinical data collected in MSpatients at Johns Hopkins University revealed a significant positivecorrelation between hippocampal NAAG concentration and patients'performances on a battery of cognitive tasks (Rahn, et al. Proc NatlAcad Sci USA, 2012. 109(49): p. 20101-6). Notably, MS patients with lowhippocampal NAAG levels showed cognitive impairment while patients withhigher levels of hippocampal NAAG exhibited normal cognition. Insupport, polymorphisms of mGluR3 have recently been linked todifferential cognitive abilities (Jablensky, et al. Genes Brain Behav,2011. 10(4): p. 410-7; Egan, et al. Proc Natl Acad Sci USA, 2004.101(34): p. 12604-9; 8 Harrison, et al. J Psychopharmacol, 2008. 22(3):p. 308-22; Sartorius, et al. Neuropsychopharmacology, 2008. 33(11): p.2626-34).

The brain metallopeptidase Glutamate Carboxypeptidase II (GCPII)catabolizes NAAG in vivo. One of the most potent (IC₅₀=300 pM) andselective GCPII inhibitors, termed 2-PMPA, has been shown tosignificantly increase brain NAAG levels and improve cognition inpreclinical models (Olszewski, et al., Transl Psychiatry, 2012. 2: p.e145; Yamada, et al. Mol Pain, 2012. 8: p. 67; Janczura, et al., Eur JPharmacol, 2013. 701(1-3): p. 27-32; Gurkoff, et al. Brain Res, 2013.1515: p. 98-107) including MS (Rahn, et al. Proc Natl Acad Sci USA,2012. 109(49): p. 20101-6). To our knowledge, 2-PMPA is the first andonly treatment strategy that has been shown to attenuate cognitiveimpairment in a preclinical model of MS. However, 2-PMPA is a polarbisphosphonate-based compound which is active only after systemic dosing(i.p. or i.v.). It has negligible oral bioavailability and is thereforeunsuitable for daily chronic dosing in patients. Using prodrugstrategies proven successful in enhancing the oral bioavailability ofother bisphosphonate compounds which are now marketed and widely used(ADEFOVIR™ and TENOFOVIR™) (Cundy, et al. J Pharm Sci, 1997. 86(12): p.1334-8; Deeks, et al., J Infect Dis, 1997. 176(6): p. 1517-23; Kearney,et al. Clin Pharmacokinet, 2004. 43(9): p. 595-612), novel orallybioavailable prodrugs of 2-PMPA can be synthesized. The presentlydisclosed subject matter provides one such prodrug, with >100-foldincrease in bioavailability in dogs, respectively clearly demonstratingfeasibility of the approach. Employing an iterative medicinal chemistryand drug metabolism/pharmacokinetic approach, it is proposed tosystematically optimize novel prodrugs with the goal of developing an,ultimately, clinical investigation in MS patients.

Prodrugs will be evaluated in an experimental autoimmuneencephalomyelitis (EAE) mouse model of multiple sclerosis. Mice will beimmunized and receive daily p.o. dosing of either vehicle or 2-PMPAprodrug from the time of immunization until sacrifice (preventionparadigm) or will be treated either with vehicle or 2-PMPA prodrug(treatment paradigm). The development and progression of the resultingdeficits will be tracked by EAE disease scores, body weightmeasurements, and cognitive testing. Post-mortem analysis of brain NAAG,2-PMPA and GCPII inhibition confirming target engagement will beperformed in tandem.

NAAG is an mGluR3 agonist which is inactivated by GCPII: N-acetylaspartyl glutamate (NAAG), one of the most abundant neuropeptides in themammalian central nervous system (CNS) (Neale, et al. J Neurochem, 2000.75(2): p. 443-52), is a selective agonist at metabotropic glutamatereceptor 3 (mGluR3) (Olszewski, et al. Schizophr Res, 2012. 136(1-3): p.160-1). As with other neurotransmitter/modulators, the concentration ofextracellular NAAG is tightly regulated. A 94 kD class II membrane boundzinc metalloenzyme termed glutamate carboxypeptidase II (GCPII, alsocalled NAALADase or NAAG peptidase) degrades into N-acetylaspartate(NAA) and glutamate (FIG. 26).

Decreased brain NAAG associated with cognitive impairment: Human studiesspanning two decades report that CNS NAAG concentrations are altered inneurological diseases with comorbid cognitive impairment (Jaarsma, etal. J Neurol Sci, 1994. 127(2): p. 230-3; Rowland, et al., GABA, andNAAG in schizophrenia. Schizophr Bull, 2013. 39(5): p. 1096-104; Tsai,et al., CNS. Brain Res, 1991. 556(1): p. 151-6), including MS (Rahn, etal. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6). Historicallypost-mortem immunohistochemical or HPLC/MS techniques were required forquantitation of brain NAAG levels, however with the recent developmentof advanced neuroimaging techniques and increased MRI magnet strength(≥3T), in vivo imaging of NAAG is now possible. Recent clinical datacollected at the Johns Hopkins hospital demonstrate a significant andselective positive correlation between hippocampal NAAG concentration inMS patients and their performance on a battery of cognitive tasks (Rahn,et al. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6). Specifically,MS patients with cognitive impairment have low hippocampal NAAG levelswhile MS patients with normal cognition have higher levels ofhippocampal NAAG (FIG. 27).

No clinically available GCPII inhibitor to date: Unfortunately, to dateno GCPII inhibitor has high potential for clinical translation. Eisai,Inc (formerly Guilford Pharmaceuticals) developed an orallybioavailable, thiol-based GCPII inhibitor which completed 2 Phase 1studies. Although the inhibitor was well-tolerated in Phase 1 (REF)subsequent immunological toxicities observed in GLP primate studieshalted its development. Importantly the toxicity was not due to theGCPII mechanism, but rather due to the thiol moiety in the compound. Asa class, thiol drugs have a risk of inducing hypersensitivity reactions(REF). Given the large unmet medical need for, second generationnon-thiol GCPII inhibitors devoid of this immunological risk foradvancement into clinical development can be synthesized. Beyond thiolinhibitors, the most potent, selective, and efficacy inhibitors of GCPIIdescribed are phosphonic acid based, however they have minimal orallybioavailability.

2-PMPA increases brain NAAG and prevents cognitive deficits in a mousemodel: The metabolite NAAG is broken down by the enzyme GCPII. Withoutwishing to be bound to any one particular theory, it is thought thatadministration of 2-phosponomethyl pentanedioic acid (2-PMPA), a potentand selective inhibitor of GCPII, would reverse cognitive impairment inan animal model of MS with known learning and memory deficits (Ziehn, etal. EAE. Lab Invest, 2010. 90(5): p. 774-86). Mice (n=10) were immunizedfor EAE, injections of 2-PMPA were administered daily from the time ofdisease induction, and behavior tests were conducted after chronicphysical signs of disease were established. While no difference inphysical severity was observed, cognition was significantly improved inmice treated with the GCPII inhibitor (EAE+2-PMPA) compared tovehicle-treated controls (EAE+Vehicle) as measured by Barnes maze (acircular land maze analogous to the Morris water maze that is used foranimals with physical disabilities) and fear conditioning tests (Rahn,et al. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6). EAE+Vehiclemice had higher Barnes maze path efficiency delta and significantlydecreased total latency delta as compared to Control+Vehicle (P<0.05),indicating cognitive impairment. Conversely, the total latency and pathefficiency of EAE+2-PMPA mice did not differ from Control+Vehicle mice.Furthermore, EAE+2-PMPA mice had significantly improved (i.e. over2-fold) path efficiency and total latency as compared to EAE+Vehiclemice (FIG. 28A and FIG. 28B, P<0.01 and P<0.05, respectively). Fearconditioning tests demonstrated a significant difference between fearmemory in EAE+2-PMPA mice compared to EAE+Vehicle mice (P<0.05). Postmortem analysis demonstrated a significant increase in brain NAAG inEAE+2-PMPA mice versus EAE+Vehicle mice (FIG. 28C, P<0.05). Takentogether, these data demonstrate that GCPII inhibition restores thecognitive and biological deficits resulting from EAE.

Conduct prodrug efficacy studies in a mouse model of multiple sclerosis:Prodrugs of 2-PMPA will be tested for in vivo efficacy. Preclinicalstudies will be conducted using the research design in which dailyintraperitoneal injection of the GCPII inhibitor 2-PMPA demonstratedsignificant beneficial effects on cognitive function (Rahn, et al. ProcNatl Acad Sci USA, 2012. 109(49): p. 20101-6). Mice will be immunizedfor chronic EAE as previously described (Rahn, et al. Proc Natl Acad SciUSA, 2012. 109(49): p. 20101-6), and daily oral dosing of prodrug orvehicle will begin from the time of immunization and continue untilsacrifice. Control groups not immunized for EAE will be included in todetermine if daily 2-PMPA prodrug administration improves learning andmemory in healthy non-EAE control mice. An EAE+2-PMPA i.p. control groupwill be included to determine if oral prodrug treatment is more or lessefficacious versus daily intraperitoneal injections 2-PMPA. Animals willbe divided into five groups (n=15/group):

-   -   Group 1=Control+Vehicle    -   Group 2=Control+2-PMPA Prodrug    -   Group 3=EAE+Vehicle    -   Group 4=EAE+2-PMPA Prodrug    -   Group 5=EAE+2-PMPA (i.p.)

Mice will be monitored daily for signs of EAE. Approximately two weeksafter disease onset, mice will be subjected to elevated plus mazetesting, followed by Barnes maze testing, then fear conditioning. Uponcompletion of the tests (approximately Day 50), animals will besacrificed and brains will be dissected. NAA, NAAG and 2-PMPA levelswill be measured in the hippocampus, cerebellum, and frontal lobe viamass spectrometry. To measure prodrug bioavailability and the effects ofGCPII inhibition on brain NAAG, five satellite animals will besacrificed prior to behavior testing (approximately 4 weekspost-immunization). The remaining 10 animals will be sacrificedfollowing the completion of all behavioral tests. The above tests willbe conducted for 2-PMPA prodrugs.

Expected Results: The GCPII inhibitor prodrug is thought to be equallyefficacious at preventing cognitive impairment in EAE as compared todaily intraperitoneal injections of 2-PMPA. EAE mice treated with the2-PMPA prodrug are expected to perform as well as Control (i.e. non-EAE)mice on Barnes maze and fear conditioning tests. Elevated plus mazeperformance, a measure of anxiety, is not expected to differ between EAEand Control cohorts. It is expected that the prodrug treatment willrestore brain NAAG levels equivalent to those observed in Control mice(Rahn, et al. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6).Previous work from our laboratory and others has demonstrated that GCPIIinhibition has no effect on cognitive function in Control mice.Therefore, Groups 1 and 2 are not expected to differ with regard tocognitive function. It is possible, however, that the improvedbioavailability of the prodrug will cause cognitive enhancing effects innormal mice.

Summary: Despite the fact that over 200,000 MS patients suffer from sometype of cognitive impairment in the United States alone, no therapieshave been developed to treat MS-associated learning and memory deficits.The failure of clinical trials designed to translate pre-existingtherapies for other neurological diseases with comorbid cognitiveimpairment, such as memantine, rivastigmine, and donepezil inAlzheimer's disease, to treatments for cognitive impairment in MSsuggest that alternative and specific treatment pathways should beexplored. To our knowledge the present approach is the first to developa drug treatment that selectively targets an established biologicaldeficit in cognitively impaired MS patients (i.e. the reduction in brainNAAG). Thus, completion of the presently disclosed studies could lead tothe first treatment specifically developed to treat cognitive impairmentin MS. NMSS thought that our NAAG/GCPII efficacy data were sufficientlyimportant to fund mechanistic research (PI: Dr. Adam Kaplin) into theaction of 2-PMPA utilizing GCPII and mGluR3 KO mice, and pharmacologicalreceptor antagonists. While related to Dr. Kaplin's project, thisindependent project is a logical and translationally-focusedcontinuation of the funded work, as 2-PMPA is not an effective long-termtreatment strategy in humans. The presently disclosed studies arerequired to develop a compound that is safe and effective for human use.

In addition to a novel treatment for cognitive impairment, these studiesmay also lead to the development of a human biomarker with clinical andtreatment applications. Recent advances in magnetic resonancespectroscopy allow for the quantitation of CNS NAAG levels in humansusing 3T or 7T MRI scanners. NAAG, therefore, can be used as a diseasebiomarker to measure changes in NAAG levels in MS patients over time,identify the ˜1 million patients who would benefit from our treatmentstrategy, and monitor drug effects over time or may be susceptible tocognitive impairment.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art. In case of aconflict between the specification and any of the incorporatedreferences, the specification (including any amendments thereof, whichmay be based on an incorporated reference), shall control. Standardart-accepted meanings of terms are used herein unless indicatedotherwise. Standard abbreviations for various terms are used herein.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

-   Alex, P.; Zachos, N. C.; Nguyen, T.; Gonzales, L.; Chen, T. E.;    Conklin, L. S.; Centola, M.; Li, X. Distinct cytokine patterns    identified from multiplex profiles of murine DSS and TNBS-induced    colitis. Inflamm. Bowel Dis. 2009, 15:341-352.-   Alex, P.; Ye, M.; Zachos, N. Z.; Sipes, J.; Nguyen, T.; Suhodrev,    M.; Gonzales, L.; Arora, Z.; Zhang, T.; Centola, M.; Guggino, S. E.;    Li, X. Clc-5 Knockout mice exhibit novel immunomodulatory effects    and are more susceptible to dextran sulphate sodium induced    colitis. J. Immunol. 2010, 184:3988-3996.-   Barditch-Crovo, P.; Deeks, S. G.; Collier, A.; Safrin, S.;    Coakley, D. F.; Miller, M.; Kearney, B. P.; Coleman, R. L.; Limy,    PAD.; Kahn, J. O.; McGowan, I.; Lietman, P. S. Phase i/ii trial of    the pharmacokinetics, safety, and antiretroviral activity of    tenofovir disoproxil fumarate in human immunodeficiency    virus-infected adults. Antimicrob. Agents Chemother. 2001,    45:2733-2739.-   Barditch-Crovo, P.; Toole, J.; Hendrix, C. W.; Cundy, K. C.;    Ebeling, D.; Jaffe, H. S.; Lietman, P. S. Anti-human    immunodeficiency virus (HIV) activity, safety, and pharmacokinetics    of adefovir dipivoxil    (9-[2-(bis-pivaloyloxymethyl)-phosphonylmethoxyethyl]adenine) in    HIV-infected patients. J. Infect. Dis. 1997, 176:406-413.-   Colombel, J. F.; Sandborn, W. J.; Rutgeerts, P.; Enns, R.;    Hanauer, S. B.; Panaccione, R.; Schreiber, S.; Byczkowski, D.; Li,    J.; Kent, J. D.; Pollack, P. F., Adalimumab for maintenance of    clinical response and remission in patients with Crohn's disease:    the CHARM trial. Gastroenterology 2007, 132:52-65.-   Cundy, K. C.; Sue, I. L.; Visor, G. C.; Marshburn, J.; Nakamura, C.;    Lee, W. A.; Shaw, J. P. Oral formulations of adefovir dipivoxil: in    vitro dissolution and in vivo bioavailability in dogs. J. Pharm.    Sci. 1997, 86:1334-1338.-   Hamilton, M. J.; Snapper, S. B.; Blumberg, R. S., Update on biologic    pathways in inflammatory bowel disease and their therapeutic    relevance. J. Gastroenterol. 2012, 47:1-8.-   Hanauer, S. B.; Feagan, B. G.; Lichtenstein, G. R.; Mayer, L. F.;    Schreiber, S.; Colombel, J. F.; Rachmilewitz, D.; Wolf, D. C.;    Olson, A.; Bao, W.; Rutgeerts, P., Maintenance infliximab for    Crohn's disease: the ACCENT I randomised trial. Lancet 2002,    359:1541-1549.-   Kaser, A., Zeissig, S., Blumberg, R. S., Inflammatory bowel disease.    Annu. Rev. Immunol. 2010, 28:573-621.-   Kozuch, P. L. and Hanauer, S. B., Treatment of inflammatory bowel    disease: A review of medical therapy. World J. Gastroenterol. 2008,    14:354-377.-   Lawrance, I. C. What is left when anti-tumour necrosis factor    therapy in inflammatory bowel diseases fails? World J.    Gastroenterol. 2014, 20:1248-1258.-   Mesters, J. R.; Barinka, C.; Li, W.; Tsukamoto, T.; Majer, P.;    Slusher, B. S.; Konvalinka, J.; Hilgenfeld, R., Structure of    glutamate carboxypeptidase II, a drug target in neuronal damage and    prostate cancer. EMBO J. 2006, 25:1375-1384.-   Rais, R.; Rojas, C.; Wozniak, K.; Wu, Y.; Zhao, M.; Tsukamoto, T.;    Rudek, M. A.; Slusher, B. S., Bioanalytical method for evaluating    the pharmacokinetics of the GCP-II inhibitor 2-phosphonomethyl    pentanedioic acid (2-PMPA). J. Pharm. Biomed. Anal. 2014,    88:162-169.-   Regueiro, M.; Siemanowski, B.; Kip, K. E.; Plevy, S., Infliximab    dose intensification in Crohn's disease. Inflamm. Bowel Dis. 2007,    13:1093-1099.-   Ristau, B. T.; O'Keefe, D. S.; Bacich, D. J., The prostate-specific    membrane antigen: Lessons and current clinical implications from 20    years of research. Urol. Oncol. 2013, 32(3):272-9.-   Sartor, R. B., Mechanisms of disease: pathogenesis of Crohn's    disease and ulcerative colitis. Nat. Clin. Pract. Gastroenterol.    Hepatol. 2006, 3:390-407.-   Schreiber, S.; Khaliq-Kareemi, M.; Lawrance, I. C.; Thomsen, O. O.;    Hanauer, S. B.; McColm, J.; Bloomfield, R.; Sandborn, W. J.,    Maintenance therapy with certolizumab pegol for Crohn's disease. N.    Engl. J. Med. 2007, 357:239-250.-   Schmidt, C.; Giese, T.; Hermann, E.; Zeuzem, S.; Meuer, S. C.;    Stallmach, A., Predictive value of mucosal TNF-alpha transcripts in    steroid-refractory Crohn's disease patients receiving intensive    immunosuppressive therapy. Inflamm. Bowel Dis. 2007, 13:65-70.-   Slusher, B. S.; Rojas, C.; Coyle, J. T., Glutamate    Carboxypeptidase II. In: Rawlings and Salvesen, editors. Handbook    for Proteolytic Enzymes, Academic Press. 3rd Edition. 2013,    1620-1626.-   Strober, W.; Fuss, I.; and Mannon, P., The fundamental basis of    inflammatory bowel disease. J. Clin. Invest. 2007, 117:514-521.-   Thackaberry, E. A. Vehicle selection for nonclinical oral safety    studies. Expert Opin. Drug Metab. Toxicol. 2013, 9:1635-1646.-   Van, A. G.; Van, R. M.; Sciot, R.; Dubois, B.; Vermeire, S.; Noman,    M.; Verbeeck, J.; Geboe, s K.; Robberecht, W.; Rutgeerts, P.,    Progressive multifocal leukoencephalopathy after natalizumab therapy    for Crohn's disease. N. Engl. J. Med. 2005, 353:362-368.-   Villablanca, E. J.; De, C. J.; Torregrosa, P. P.; Cassani, B.;    Nguyen, D. D.; Gabrielsson, S.; Mora, J. R. beta7 integrins are    required to give rise to intestinal mononuclear phagocytes with    tolerogenic potential. Gut 2013, Sep. 12.-   Xavier, R. J. and Podolsky, D. K., Unravelling the pathogenesis of    inflammatory bowel disease. Nature 2007, 448:427-434.-   Zhang, T.; Song, B.; Zhu, W.; Xu, X.; Gong, Q. Q.; Morando, C.;    Dassopoulos, T.; Newberry, R. D.; Hunt, S. R.; Li, E., An ileal    Crohn's disease gene signature based on whole human genome    expression profiles of disease unaffected ileal mucosal biopsies.    PLoS ONE 2012; 7:e37139.-   Dutta, R. and B. D. Trapp, Pathogenesis of axonal and neuronal    damage in multiple sclerosis. Neurology, 2007. 68(22 Suppl 3): p.    S22-31; discussion S43-54.-   Calabrese, M., et al., Cortical lesions and atrophy associated with    cognitiveimpairment in relapsing-remitting multiple sclerosis. Arch    Neurol, 2009. 66(9): p. 1144-50.-   Neale, J. H., T. Bzdega, and B. Wroblewska,    N-Acetylaspartylglutamate: the most abundant peptide    neurotransmitter in the mammalian central nervous system. J    Neurochem, 2000. 75(2): p. 443-52.-   Olszewski, R. T., T. Bzdega, and J. H. Neale, mGluR3 and not mGluR2    receptors mediate the efficacy of NAAG peptidase inhibitor in    validated model of schizophrenia. Schizophr Res, 2012. 136(1-3): p.    160-1.-   Rahn, K. A., et al., Inhibition of glutamate carboxypeptidase II    (GCPII) activity as a treatment for cognitive impairment in multiple    sclerosis. Proc Natl Acad Sci USA, 2012. 109(49): p. 20101-6.-   Jablensky, A., et al., Polymorphisms associated with normal memory    variation also affect memory impairment in schizophrenia. Genes    Brain Behav, 2011. 10(4): p. 410-7.-   Egan, M. F., et al., Variation in GRM3 affects cognition, prefrontal    glutamate, and risk for schizophrenia. Proc Natl Acad Sci USA, 2004.    101(34): p. 12604-9.-   Harrison, P. J., et al., The group II metabotropic glutamate    receptor 3 (mGluR3, mGlu3, GRM3): expression, function and    involvement in schizophrenia. J Psychopharmacol, 2008. 22(3): p.    308-22.-   Sartorius, L. J., et al., Expression of a GRM3 splice variant is    increased in the dorsolateral prefrontal cortex of individuals    carrying a schizophrenia risk SNP. Neuropsychopharmacology, 2008.    33(11): p. 2626-34.-   Olszewski, R. T., et al., NAAG peptidase inhibitors block cognitive    deficit induced by MK-801 and motor activation induced by    d-amphetamine in animal models of schizophrenia. Transl    Psychiatry, 2012. 2: p. e145.-   Yamada, T., et al., NAAG peptidase inhibition in the periaqueductal    gray and rostral ventromedial medulla reduces flinching in the    formalin model of inflammation. Mol Pain, 2012. 8: p. 67.-   Janczura, K. J., et al., NAAG peptidase inhibitors and deletion of    NAAG peptidase gene enhance memory in novel object recognition test.    Eur J Pharmacol, 2013. 701(1-3): p. 27-32.-   Gurkoff, G. G., et al., NAAG peptidase inhibitor improves motor    function and reduces cognitive dysfunction in a model of TBI with    secondary hypoxia. Brain Res, 2013. 1515: p. 98-107.-   Cundy, K. C., et al., Oral formulations of adefovir dipivoxil: in    vitro dissolution and in vivo bioavailability in dogs. J Pharm    Sci, 1997. 86(12): p. 1334-8.-   Deeks, S. G., et al., The safety and efficacy of adefovir dipivoxil,    a novel anti-human immunodeficiency virus (HIV) therapy, in    HIV-infected adults: a randomized, double-blind, placebo-controlled    trial. J Infect Dis, 1997. 176(6): p. 1517-23.-   Kearney, B. P., J. F. Flaherty, and J. Shah, Tenofovir disoproxil    fumarate: clinical pharmacology and pharmacokinetics. Clin    Pharmacokinet, 2004. 43(9): p. 595-612.-   Jaarsma, D., L. Veenma-van der Duin, and J. Korf, N-acetylaspartate    and N-acetylaspartylglutamate levels in Alzheimer's disease    post-mortem brain tissue. J Neurol Sci, 1994. 127(2): p. 230-3.-   Rowland, L. M., et al., In vivo measurements of glutamate, GABA, and    NAAG in schizophrenia. Schizophr Bull, 2013. 39(5): p. 1096-104.-   Tsai, G. C., et al., Reductions in acidic amino acids and    N-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS.    Brain Res, 1991. 556(1): p. 151-6.-   Ziehn, M. O., et al., Hippocampal CA1 atrophy and synaptic loss    during experimental autoimmune encephalomyelitis, EAE. Lab    Invest, 2010. 90(5): p. 774-86.-   Jackson, P. F., et al., Design and pharmacological activity of    phosphinic acid based NAALADase inhibitors. J Med Chem, 2001.    44(24): p. 4170-5.-   Jackson, P. F., et al., Design, synthesis, and biological activity    of a potent inhibitor of the neuropeptidase N-acetylated    alpha-linked acidic dipeptidase. J Med Chem, 1996. 39(2): p. 619-22.-   Slusher, B. S., et al., Selective inhibition of NAALADase, which    converts NAAG to glutamate, reduces ischemic brain injury. Nat    Med, 1999. 5(12): p. 1396-402.-   Rais, R., et al., Bioanalytical method for evaluating the    pharmacokinetics of the GCP-II inhibitor 2-phosphonomethyl    pentanedioic acid (2-PMPA). J Pharm Biomed Anal, 2013. 88: p. 162-9.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A composition comprising a dose of a compoundof formula (I) or formula (II) dissolved in an aqueous solution, whereinthe aqueous solution comprises a pharmaceutically acceptable carrier anda physiologically compatible buffer, and wherein the compound of formula(I) or formula (II) are:

wherein: (a) each R₁ is H; each R₂ is selected from the group consistingof H, alkyl, Ar, —(CR₅R₆)_(n)—Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇,—(CR₅R₆)_(n)—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; each R₃ is selected from the group consistingof H, alkyl, Ar, —(CR₅R₆)_(n)—Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇,—(CR₅R₆)_(n)—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; and each R₄ is selected from the groupconsisting of —(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,—(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; (b) each R₁ is alkyl; each R₂ is selected fromthe group consisting of H, alkyl, Ar, —(CR₅R₆)_(n)—Ar,—(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,—(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; each R₃ is selected from the group consistingof H, alkyl, Ar, —(CR₅R₆)_(n)—Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇,—(CR₅R₆)_(n)—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; and each R₄ is selected from the groupconsisting of Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,—(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; (c) each R₁ is —(CR₅R₆)_(n)—Ar; each R₂ isselected from the group consisting of H, alkyl, Ar, —(CR₅R₆)_(n)—Ar,—(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,—(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; each R₃ is selected from the group consistingof H, alkyl, Ar, —(CR₅R₆)_(n)—Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇,—(CR₅R₆)_(n)—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; and each R₄ is selected from the groupconsisting of Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,—(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; or (d) each R₁ is selected from Ar,—(CR₅R₆)_(n)—O—C(═O)—R₇, —(CR₅R₆)_(n)—C(═O)—O—R₇,—(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; each R₂ is selected from the group consistingof H, alkyl, Ar, —(CR₅R₆)_(n)—Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇,—(CR₅R₆)_(n)—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; each R₃ is selected from the group consistingof H, alkyl, Ar, —(CR₅R₆)_(n)—Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇,—(CR₅R₆)_(n)—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; and each R₄ is selected from the groupconsisting of H, alkyl, Ar, —(CR₅R₆)_(n)—Ar, —(CR₅R₆)_(n)—O—C(═O)—R₇,—(CR₅R₆)_(n)—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—C(═O)—O—R₇, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—NR₈R₉, and—(CR₅R₆)_(n)—C(═O)—NR₈R₉; wherein: each n is an integer from 1 to 20;each m is an integer from 1 to 20; each R₅ and R₆ is independentlyselected from the group consisting of H, alkyl, and alkylaryl; each R₇is independently straight chain or branched alkyl; each Ar is aryl,substituted aryl, heteroaryl or substituted heteroaryl; each R₈ and R₉are independently H or alkyl; and each R_(3′) and R_(4′) areindependently H or alkyl; or pharmaceutically acceptable salts thereof.2. The composition of claim 1, wherein the compound is a compound offormula (I) and: R₁ is H; R₂ and R₃ are each selected from the groupconsisting of H, —(CR₅R₆)_(n)—O—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—(CR₅R₆)_(n)—O—C(═O)—R₇, —Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, and—(CR₅R₆)_(n)—O—C(═O)—O—R₇; and R₄ is selected from the group consistingof —(CR₅R₆)_(n)—O—R₇, —(CR₅R₆)_(n)—Ar—O—C(═O)—R₇,—Ar—C(═O)—O—(CR₅R₆)_(n)—R₇, —(CR₅R₆)_(n)—O—C(═O)—R₇ and—(CR₅R₆)_(n)—O—C(═O)—O—R₇; or pharmaceutically acceptable salts thereof.3. The composition of claim 1, wherein the compound is a compound offormula (I) and: R₁ is alkyl; R₂ and R₃ are each independently selectedfrom the group consisting of H, alkyl, —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—Ar—O—C(═O)—R₇, —(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇,—(CR₅R₆)_(n)—O—C(═O)—R₇ and —(CR₅R₆)_(n)—O—C(═O)—O—R₇; and R₄ isselected from the group consisting of —(CR₅R₆)_(n)—O—R₇,—(CR₅R₆)_(n)—Ar—O—C(═O)—R₇, —(CR₅R₆)_(n)—O—[(CR₅R₆)_(n)—O]_(m)—R₇,—(CR₅R₆)_(n)—O—C(═O)—R₇ and —(CR₅R₆)_(n)—O—C(═O)—O—R₇; orpharmaceutically acceptable salts thereof.
 4. The composition of claim1, wherein the compound is a compound of formula (I) and: R₁ is selectedfrom —(CR₅R₆)_(n)—O—C(═O)—R₇ and —(CR₅R₆)_(n)—O—C(═O)—O—R₇; and R₂ R₃,and R₄ are each independently selected from H, Ar,—(CR₅R₆)_(n)—O—C(═O)—R₇, and —(CR₅R₆)_(n)—O—C(═O)—O—R₇; orpharmaceutically acceptable salts thereof.
 5. The composition of claim1, wherein the compound is a compound of formula (I) and: one of R₁, R₂,R₃, or R₄ is H and the other three are each independently selected fromthe group consisting of: —(CR₅R₆)_(n)—O—C(═O)—R₇ and—(CR₅R₆)_(n)—O—C(═O)—O—R₇; wherein R₅ and R₆ are each independentlyselected from the group consisting of H, C₁₋₈ straight-chain alkyl, andC₁₋₈ branched-chain alkyl; R₇ is C₁₋₈ straight-chain alkyl, and C₁₋₈branched-chain alkyl; or pharmaceutically acceptable salts thereof. 6.The composition of claim 1, wherein the compound is a compound offormula (I) and: R₂ is H; and R₁, R₃, and R₄ are each independentlyselected from the group consisting of: —(CR₅R₆)_(n)—O—C(═O)—R₇ and—(CR₅R₆)_(n)—O—C(═O)—O—R₇; wherein R₅ and R₆ are each independentlyselected from the group consisting of H, C₁₋₈ straight-chain alkyl, andC₁₋₈ branched-chain alkyl; R₇ is C₁₋₈ straight-chain alkyl or C₁₋₈branched-chain alkyl; or pharmaceutically acceptable salts thereof. 7.The composition of claim 6, wherein R₅ and R₆ are each H.
 8. Thecomposition of claim 1, wherein the compound of formula (I) is selectedfrom the group consisting of:

or pharmaceutically acceptable salts thereof.
 9. The composition ofclaim 1, wherein the pharmaceutically acceptable carrier comprisesbuffered saline.
 10. The composition of claim 1, wherein thepharmaceutically acceptable carrier comprises Hank's solution.
 11. Thecomposition of claim 1, wherein the pharmaceutically acceptable carriercomprises Ringer's solution.
 12. The composition of claim 1, wherein thedose of the compound of formula (I) or formula (II) has a range fromabout 1 mg to about 50 mg.
 13. The composition of claim 1, wherein thedose of the compound of formula (I) or formula (II) has a range fromabout 5 mg to about 40 mg.
 14. The composition of claim 1, wherein thedose of the compound of formula (I) or formula (II) has a range fromabout 10 mg to about 30 mg.
 15. The composition of claim 14, wherein thedose of the compound of formula (I) or formula (II) is about 10 mg. 16.A method for treating a disease or condition, the method comprisingadministering to a subject in need of treatment thereof, a compositionof claim 1 comprising a dose of a compound of formula (I) or formula(II) dissolved in an aqueous solution, wherein the aqueous solutioncomprises a pharmaceutically acceptable carrier and a physiologicallycompatible buffer.
 17. The method of claim 16, wherein the dose isadministered more than once in a timed-form.
 18. The method of claim 16,wherein the dose is administered in combination with one or moreadditional therapeutic agents.
 19. The method of claim 16, wherein thedisease or condition comprises colon cancer or prostate cancer.